Ophthalmic compositions

ABSTRACT

The embodiments disclosed herein relate to ophthalmic compositions comprising calcineurin inhibitors or mTOR inhibitors, and more particularly to methods for treating an ocular disease and/or condition using the disclosed compositions. According to aspects illustrated herein, there is provided a pharmaceutical composition that includes a calcineurin inhibitor or an mTOR inhibitor; a first surfactant with an HLB index greater than about 10; and a second surfactant with an HLB index of greater than about 13, wherein an absolute difference between the HLB index of the first surfactant and the HLB index of the second surfactant is greater than about 3, and wherein the composition forms mixed micelles.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/627,063, filed Feb. 20, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/974,241, filed Aug. 23, 2013, now abandoned,which is a continuation of U.S. patent application Ser. No. 13/213,451,filed Aug. 19, 2011, now U.S. Pat. No. 8,535,694, which is a divisionalapplication of U.S. patent application Ser. No. 12/247,701, filed Oct.8, 2008, now U.S. Pat. No. 8,435,544, which claims the benefit of andpriority to U.S. Provisional Application No. 60/997,796, filed Oct. 8,2007, U.S. Provisional Application No. 60/992,205, filed Dec. 4, 2007,U.S. Provisional Application No. 61/038,223, filed Mar. 20, 2008, andU.S. Provisional Application No. 61/099,420, filed Sep. 23, 2008, thedisclosures of each of these applications are hereby incorporated hereinby reference in their entirety.

FIELD

The embodiments disclosed herein relate to stable ophthalmiccompositions comprising calcineurin inhibitors or mTOR inhibitors, andmore particularly to methods for treating an ocular disease and/orcondition using the disclosed compositions.

BACKGROUND

Disease and injury to the anterior surface of the eye are the leadingcauses of visits to physicians for medical eye care in the UnitedStates. These diseases and injuries rank among the most painful of eyeconditions and can lead to disability and blindness. Major clinicalproblems of the surface of the eye include ocular surface drying, tearfilm abnormalities, and related complications; ocular surface woundswith resultant pathology and scarring; corneal dysfunction dystrophiesand inherited disease; inflammatory disease; and external ocularinfections. Eye diseases and injuries can have symptoms ranging fromitchy, runny eyes to impaired vision. Therefore, it is important toaddress eye problems right away, as some diseases can progressivelyworsen or even trigger other serious problems. Most pharmacologicmanagement of ocular disease includes the topical application ofsolutions to the surface of the eye as drops. Despite the relativelysmall proportion of a topically applied drug dose that ultimatelyreaches anterior segment ocular tissues, topical formulations remaineffective, largely because of the very high concentrations of drugs thatare administered.

Disease and injury to tissues of the posterior segment of the eye,including the retina and choroid, is involved in many of the most commonblinding diseases in the industrialized world. Age-related maculardegeneration (AMD) alone impacts more than 10 million Americans. Severevision loss from AMD and other diseases affecting the posterior segment,including diabetic retinopathy, glaucoma, and retinitis pigmentosaaccounts for most cases of irreversible blindness world wide. Currently,the treatment of posterior segment disease is to a significant extentlimited by the difficulty in delivering effective doses of drugs totarget tissues in the posterior eye.

SUMMARY

Ophthalmic compositions comprising calcineurin inhibitors or mTORinhibitors are disclosed herein. The ophthalmic compositions of thepresent disclosure are aqueous solutions of mixed micelles. Theophthalmic compositions disclosed herein are biocompatible, and areparticularly useful for topical application to the eye for the treatmentof an eye condition. According to aspects illustrated herein, there isprovided a pharmaceutical composition that includes a calcineurininhibitor or an mTOR inhibitor; a first surfactant with an HLB indexgreater than about 10; and a second surfactant with an HLB index ofgreater than about 13, wherein an absolute difference between the HLBindex of the first surfactant and the HLB index of the second surfactantis greater than about 3, and wherein the composition forms mixedmicelles.

According to aspects illustrated herein, there is provided apharmaceutical composition that includes a calcineurin inhibitor;vitamin E TPGS; and octoxynol 40, wherein the composition is suitablefor topical application to ocular tissue.

According to aspects illustrated herein, there is provided apharmaceutical composition that includes an mTOR inhibitor; vitamin ETPGS; and octoxynol 40, wherein the composition is suitable for topicalapplication to ocular tissue.

According to aspects illustrated herein, there is provided a method ofpreparing a mixed micelle composition that includes mixing a calcineurininhibitor or a mTOR inhibitor with a first surfactant having an HLBindex greater than about 10 and a second surfactant having an HLB indexof greater than about 13 in a solvent to form a solvent solution;evaporating the solvent solution to form a near-solid matter; hydratingthe near-solid matter with an aqueous solution; and dissolving thenear-solid mixture to produce the mixed micelle composition, wherein thecomposition is optically clear.

According to aspects illustrated herein, there is provided a method fortreating an ocular disease in a patient in need thereof that includesadministering topically to an eye of the patient a compositioncomprising a therapeutically effective amount of a calcineurin inhibitoror mTOR inhibitor, the composition further having vitamin E TPGS andoctoxynol-40, wherein the composition is an aqueous solution of mixedmicelles.

According to aspects illustrated herein, there is provided a method fortreating, reducing, ameliorating, or alleviating an inflammatory oculardisease in an animal that includes providing a mixed micellarpharmaceutical composition having a calcineurin inhibitor or an mTORinhibitor encapsulated in micelles, the micelles formed with a firstsurfactant with an HLB index greater than about 10 and a secondsurfactant with an HLB index of greater than about 13; and administeringto the animal an amount of the pharmaceutical composition at a frequencysufficient to treat, reduce, ameliorate, or alleviate the inflammatoryocular disease.

According to aspects illustrated herein, there is provided a method fortreating, reducing, ameliorating, or alleviating a back-of-the-eyecondition or disorder in a subject that includes providing a mixedmicellar pharmaceutical composition having a calcineurin inhibitorencapsulated in micelles formed with a first surfactant with an HLBindex greater than about 10 and a second surfactant with an HLB index ofgreater than about 13; and administering to the subject an amount of thepharmaceutical composition at a frequency sufficient to treat, reduce,ameliorate, or alleviate the back-of-the-eye condition or disorder.

According to aspects illustrated herein, there is provided an artificialtear composition that includes an aqueous solution of mixed micelles,the mixed micelles formed from a vitamin E tocopherol polyethyleneglycol succinate (TPGS) derivative and an ethoxylated octylphenolsurfactant.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings, wherein like structures are referredto by like numerals throughout the several views. The drawings shown arenot necessarily to scale, with emphasis instead generally being placedupon illustrating the principles of the presently disclosed embodiments.

FIG. 1 shows a graphical representation of Mean Schirmer Tear Test (STT)values of canine KCS patients through 30 days of treatment with anembodiment of a mixed micellar formulation containing 0.2% voclosporinof the present disclosure.

FIG. 2 shows tissue levels of voclosporin after a single (1 day) topicaldose of a mixed micellar pharmaceutical composition of the presentlydisclosed embodiments having ¹⁴C-voclosporin to female New Zealand WhiteRabbits. Therapeutic levels of voclosporin were noticed even at the24-hour mark, supporting once daily (QD) dosing is possible with theaqueous mixed micellar composition of the presently disclosedembodiments. The experiment included male rabbits also with similarresult (data not shown).

FIGS. 3A-D show mean ocular tissue concentrations of voclosporin after asingle (1 day) or repeat (7 days), bilateral, once daily, topical doseof a mixed micellar pharmaceutical composition of the presentlydisclosed embodiments having ¹⁴C-voclosporin to female New Zealand WhiteRabbits. FIG. 3A shows the mean ocular tissue concentration ofvoclosporin in the cornea. FIG. 3B shows the mean ocular tissueconcentration of voclosporin in the iris/ciliary body. FIG. 3C shows themean ocular tissue concentration of voclosporin in the lacrimal gland.FIG. 3D shows the mean ocular tissue concentration of voclosporin in thelens.

FIGS. 4A-D show mean ocular tissue concentrations of voclosporin after asingle (1 day) or repeat (7 days), bilateral, once daily, topical doseof a mixed micellar pharmaceutical composition of the presentlydisclosed embodiments having ¹⁴C-voclosporin to female New Zealand WhiteRabbits. FIG. 4A shows the mean ocular tissue concentration ofvoclosporin in the lower conjunctiva. FIG. 4B shows the mean oculartissue concentration of voclosporin in the lower eyelid. FIG. 4C showsthe mean ocular tissue concentration of voclosporin in the nictitatingmembrane. FIG. 4D shows the mean ocular tissue concentration ofvoclosporin in the sclera.

FIGS. 5A-D show mean ocular tissue and fluid concentrations ofvoclosporin after a single (1 day) or repeat (7 days), bilateral, oncedaily, topical dose of a mixed micellar pharmaceutical composition ofthe presently disclosed embodiments having ¹⁴C-voclosporin to female NewZealand White Rabbits. FIG. 5A shows the mean ocular tissueconcentration of voclosporin in the upper conjunctiva. FIG. 5B shows themean ocular tissue concentration of voclosporin in the upper eyelid.FIG. 5C shows the mean ocular fluid concentration of voclosporin in theaqueous humor. FIG. 5D shows the mean ocular fluid concentration ofvoclosporin in the vitreous humor.

FIGS. 6A-D show mean ocular tissue and fluid concentrations ofvoclosporin after a single (1 day) or repeat (7 days), bilateral, oncedaily, topical dose of a mixed micellar pharmaceutical composition ofthe presently disclosed embodiments having ¹⁴C-voclosporin to female NewZealand White Rabbits. FIG. 6A shows the mean ocular fluid concentrationof voclosporin in tears. FIG. 6B shows the mean ocular tissueconcentration of voclosporin in the submandibular lymph node. FIG. 6Cshows the mean ocular tissue concentration of voclosporin in the opticnerve. FIG. 6D shows the mean ocular tissue concentration of voclosporinin the choroid/retina.

FIG. 7 is a graph showing C_(max) values of voclosporin after repeat (7day), bilateral, once daily, topical dose of a mixed micellarpharmaceutical composition of the presently disclosed embodiments having¹⁴C-voclosporin to female New Zealand White Rabbits.

While the above-identified drawings set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION

The presently disclosed embodiments are directed towards pharmaceuticalcompositions comprising calcineurin inhibitors or mTOR inhibitors in amixed micellar topical dosage form. The pharmaceutical compositions ofthe present disclosure have been found to treat, reduce, ameliorate andalleviate ocular conditions in a patient or subject. In an embodiment,the compositions can be used for the treatment of ocular diseases,including inflammatory ocular surface diseases. Examples of suchdiseases include, but are not limited to, dry eye syndrome (DES),Sjogren's syndrome, uveitis, conjunctivitis (pink eye), keratitis,keratoconjunctivitis, vernal keratoconjunctivitis (VKC), atopickeratoconjunctivitis (AKC), autoimmune disorders of the ocular surface,such as cicatrizing conjunctivitis, blepharitis, and scleritis.

In an embodiment, the compositions can be used for the treatment of aback-of-the eye condition and/or disorder. Examples of suchconditions/disorders include, but are not limited to, posterior uveitis,age-related macular degeneration (AMD, wet and dry), diabetic eyeconditions such as diabetic retinopathy (DR) and diabetic macular edema(DME), glaucoma, ocular hypertension, post-operative eye pain andinflammation, ocular neovascularization such as posterior segmentneovascularization (PSNV), proliferative vitreoretinopathy (PVR),cytomegalovirus (CMV) retinitis, choroidal neovascular membranes (CNVM),vascular occlusive diseases, retinitis pigmentosa, optic neuritis,cicatrizing ocular surface diseases, ocular infections, inflammatoryocular diseases, ocular surface diseases, corneal diseases, retinaldiseases such as epiretinal membrane, ocular manifestations of systemicdiseases, hereditary eye conditions, and ocular tumors.

In an embodiment, the compositions can be used for preventing transplantrejection of, for example, corneal allografts following transplantation.It is well known that in inflammation T-lymphocytes play a critical rolein mediating rejection of foreign tissues. Prevention of rejection is ofparamount importance in maintaining the health of transplanted corneas.Rejection may occur in any of the layers comprising the cornea, forexample, the corneal epithelium, the corneal stroma or the cornealendothelium. The functioning of the cornea can be compromised followingendothelial rejection. The endothelial layer serves to maintain thecornea in a compact state, acting as a pump by removing water from thecorneal stroma. If the function of the endothelial layer is compromised,disorientation of collagen fibers can ensue, and transparency of thecornea can be lost. Human endothelial cells are non-replicative, and asa consequence, donor cell loss in the setting of rejection isirreversible and may lead to diminished graft function and survival.Thus, the goal of either prevention or treatment of rejection in cornealtransplant recipients is to minimize endothelial cell loss. Thecompositions of the present disclosure can be used for the prevention ofrejection following corneal allograft transplantation.

A patient or subject to be treated by any of the compositions or methodsof the present disclosure can mean either a human or a non-human animal.In an embodiment, the present disclosure provides methods for thetreatment of an ocular disease in a human patient in need thereof. In anembodiment, the present disclosure provides methods for the treatment ofan inflammatory ocular disease in a human patient in need thereof. Inanother embodiment, the present disclosure provides methods for thetreatment of an ocular disease in a veterinary patient in need thereof,including, but not limited to dogs, horses, cats, rabbits, gerbils,hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, andother zoological animals.

As used herein, the terms “ocular disease,” “ocular condition,” “eyedisease,” and “eye condition” refer to diseases/conditions of the eye(s)that can be sight threatening, lead to eye discomfort, and may signalsystemic health problems.

As used herein, the term “anterior segment disease” refers to alldisorders that affect the eye surface, anterior chamber, iris andciliary body and lens of the eye. The eye surface is composed of thecornea, conjunctiva, eyelids, lacrimal and meibomian glands, and theinterconnecting nerves.

As used herein, the terms “posterior segment eye disease” and“back-of-the-eye disease” refer to all disorders that affect theposterior segment of the eye. A posterior eye disease is a disease whichprimarily affects a posterior ocular site such as choroid or sclera,vitreous, vitreous chamber, retina, optic nerve, and blood vessels andnerves which vascularize or innervate a posterior ocular site.

As used herein, the terms “biocompatible” and “nonirritating” refer tothe property of being biologically compatible by not producing a toxic,injurious or immunological response in living tissue. The compositionsof the present disclosure are biocompatible. Similarly, none of thecomponents of the compositions of the present disclosure are inherentlyirritating to ocular tissues.

As used herein, the term “emulsion” refers to a mixture of two or moreimmiscible liquids, where one liquid is dispersed in another. Anemulsion, for example, an intimate mixture of oil and water, isgenerally of a cloudy or milky appearance.

As used herein, the term “micelle” refers to an aggregate (or cluster)of surfactant molecules. Micelles only form when the concentration ofsurfactant is greater than the critical micelle concentration (CMC).Surfactants are chemicals that are amphipathic, which means that theycontain both hydrophobic and hydrophilic groups. Micelles can exist indifferent shapes, including spherical, cylindrical, and discoidal. Amicelle comprising at least two different molecular species is a mixedmicelle. The ophthalmic compositions of the present disclosure includean aqueous, clear, mixed micellar solution.

Polymeric micelles are exploited as pharmaceutical nanocarriers for thedelivery of poorly water-soluble (i.e., water-insoluble) or hydrophobicdrugs, which can be solubilized in the hydrophobic inner core of amicelle. Micelles can therefore serve to improve solubility andbioavailability of various hydrophobic drugs. The small size of micelles(typically about 10 to about 100 nm) allows for efficient accumulationof an associated active moiety into targeted tissues. Also, the smallsize of micelles allows the advantage of sterilization of micelles byfiltration through membranes with the cut off size 0.22 μm. Micelles canbe formed from one or more polymeric nonionic surfactants. Since themicelle size is smaller than visible light wavelengths, it is believedthat the light is not scattered by the small micelles resulting in atransparent, clear solution.

As used herein, the term “optical clarity” refers to 90% or greatertransmission of light of 400 nm wavelength in a 1.0 centimeter path. Theclarity of the solution results from the micelle size which is typicallysmaller than the smallest wavelength of a visible light radiation (about350 nm). In an embodiment, the ophthalmic compositions of the presentdisclosure are substantially clear with an absorption in general, below0.1; preferably with absorption, below 0.05 measured at 400 nm.

The HLB (hydrophilic/lipophilic balance) index value is a conceptintroduced by Griffin in 1950 as a measure of the hydrophilicity orlipophilicity of nonionic surfactants. It can be determinedexperimentally by the phenol titration method of Marszall; see“Parfumerie, Kosmetik”, Vol. 60, 1979, pp. 444-448; further literaturereferences can be found in Rompp, Chemistry Lexicon, 8th Edition 1983,p. 1750. See also, for example, U.S. Pat. No. 4,795,643 (Seth).

Dry eye syndrome (DES, Chronic dry eye, Keratitis sicca; Xerophthalmia;Keratoconjunctivitis sicca) can be defined as a condition that includesa variety of disorders that result in a loss of, or altered compositionof, the natural tear film, which maintains the surface of the eye.Without this tear film, vision is impaired and patients may suffersevere ocular discomfort. DES can be caused by excessive tearevaporation or by a reduction of tear production in the lacrimal gland,which is the site of tear production. Though the exact causes of thiscondition are unknown, there is evidence supporting the link betweenreduced tear production and lacrimal gland inflammation. Currentlyavailable medications for DES are leaving substantial room for moreeffective and better tolerated products.

DES may also be a symptom of Sjogren's syndrome which is an autoimmunedisorder in which the glands that produce tears and saliva aredestroyed. This leads to dry mouth, decreased tearing, and other drymucous membranes.

Uveitis is an inflammation inside the eye affecting the uvea. The uveais the layer of the eye between the sclera and the retina, and includesthe iris, ciliary body and the choroid. The uvea supplies most of theblood supply to the retina. Uveitis can be considered an autoimmunedisease resulting in chronic inflammation of the eye. There issubstantial evidence indicating the involvement of T-lymphocytes, keycells involved in inflammatory processes, in the development of uveitis.The inflammation can cause areas of scarring on the choroid and retinathat cause areas of vision loss. There are various forms of uveitisincluding anterior uveitis, pars planitis, and posterior uveitis.Serious complications may occur if uveitis is left untreated; includingcataracts, glaucoma, retinal detachment, retinal edema and permanentvision loss.

Anterior uveitis (iritis) occurs in the front of the eye and is the mostcommon form of uveitis. Par planitis is an inflammation of the parsplana, a narrow area between the iris and the choroid. This conditionoccurs more frequently in young men, but is usually not associated withanother disease. Posterior uveitis (chondroitis) affects primarily thechoroid; the back portion of the uveal tract. If the retina is alsoinvolved, it is called chorioretinitis. Posterior uveitis may occur inassociation with an autoimmune disease, or follow a systemic infection.In posterior uveitis, inflammation can last from months to years and maycause permanent vision damage, even with treatment.

Uveitis can cause vision impairment, ocular pain, and loss of vision. Itis estimated that about 10% of new cases of blindness in the U.S. arecaused by uveitis. Approximately 300,000 people suffer from uveitis inthe U.S. alone, the majority of whom are affected by anterior uveitis.The only therapeutic class approved by the FDA for treatment of uveitisis corticosteroids, which are noted for multiple side effects, such ashypertension, hyperglycemia, and hypercholesterolemia, and in the eye,glaucoma and cataract formation.

Conjunctivitis (pink eye) describes a group of diseases that causeswelling, itching, burning, and redness of the conjunctiva, theprotective membrane that lines the eyelids and covers exposed areas ofthe sclera, or white of the eye.

Keratitis is an inflammation of the cornea (clear portion in the frontof the eye). Keratitis can be caused by an infection (bacterial, fungal,viral, parasite, etc.) or a non-infectious agent (e.g., certain types ofauto-immune diseases are associated with a variety of non-infectiouskeratitises).

Keratoconjunctivitis refers to an inflammation of the cornea andconjunctiva.

Vernal keratoconjunctivitis (VKC) is a recurrent ocular inflammatorydisease characterized by hard, elevated, cobblestone like bumps on theupper eyelid. There may also be swellings and thickening of theconjunctiva. The conjunctiva is the outermost membrane which lines theeyelids as well as the exposed parts of the eye, except for the cornea.

Atopic keratoconjunctivitis is the result of a condition called atopy.Atopy is a genetic condition whereby the immune system produces higherthan normal antibodies in response to a given allergen.

Systemic immune mediated diseases such as cicatrizing conjunctivitis andother autoimmune disorders of the ocular surface represent a clinicallyheterogeneous group of conditions where acute and chronic autoreactivemechanisms can cause significant damage to the eye. When severe andaffecting the epithelium and substantia propria of the conjunctiva,cicatrization can ensue, leading to significant mechanical alterationsas a result of the fibrosis. These conditions, though generallyinfrequent, can be the cause of profound pathology and visualdisability.

Blepharitis is a common condition that causes inflammation of theeyelids.

Scleritis is a serious inflammatory disease that affects the white outercoating of the eye, known as the sclera.

Calcineurin is a calcium/calmodulin-regulated protein phosphataseinvolved in intracellular signaling. Calcineurin inhibitors aresubstances which block calcineurin dephosphorylation of appropriatesubstrates, by targeting calcineurin phosphatase (PP2B, PP3), a cellularenzyme that is involved in gene regulation. Another class of compoundsthat exhibit this general therapeutic profile are the mTOR inhibitors.mTOR inhibitors target a molecular target known as “mammalian target ofrapamycin” (mTOR). A prototypical compound of this class is sirolimus.

Age-related macular degeneration (AMD) is a disease associated withaging that gradually destroys sharp, central vision. AMD affects themacula, which is located at the center of the retina. AMD occurs in twoforms: wet and dry. Wet AMD occurs when abnormal blood vessels behindthe retina start to grow under the macula. These new blood vessels tendto be very fragile and often leak blood and fluid. The blood and fluidraise the macula from its normal place at the back of the eye. Damage tothe macula occurs rapidly. Dry AMD occurs when the light-sensitive cellsin the macula slowly break down, gradually blurring central vision inthe affected eye.

Diabetes can affect the eye in a number of ways. Diabetic retinopathy(DR) is a complication of diabetes that results from damage to the bloodvessels of the light-sensitive tissue at the back of the eye (theretina). At first, diabetic retinopathy may cause no symptoms or onlymild vision problems. Eventually, however, diabetic retinopathy canresult in blindness. Diabetic macular edema (DME) is the swelling of theretina in diabetes mellitus due to leaking of fluid from blood vesselswithin the macula.

Ocular neovascularization is the abnormal or excessive formation ofblood vessels in the eye. Ocular neovascularization has been shown indiabetic retinopathy and age-related macular degeneration (ARMD).

Proliferative vitreoretinopathy (PVR) is scar tissue formation withinthe eye. “Proliferative” because cells proliferate and“vitreoretinopathy” because the problems involve the vitreous andretina. In PVR scar tissue forms in sheets on the retina which contract.This marked contraction pulls the retina toward the center of the eyeand detaches and distorts the retina severely. PVR can occur bothposteriorly and anteriorly with folding of the retina both anteriorlyand circumferentially.

The cytomegalovirus (CMV) is related to the herpes virus and is presentin almost everyone. When a person's immune system is suppressed becauseof disease (HIV), organ or bone marrow transplant, or chemotherapy, theCMV virus can cause damage and disease to the eye and the rest of thebody. CMV affects the eye in about 30% of the cases by causing damage tothe retina. This is called CMV retinitis.

Optic neuritis occurs when the optic nerve becomes inflamed and themyelin sheath becomes damaged or is destroyed. Nerve damage that occursin the section of the optic nerve located behind the eye, is calledretrobulbar neuritis, which is another term sometimes used for opticneuritis.

Also known as macular pucker, epiretinal membrane is a scar-tissue likemembrane that forms over the macula. It typically progresses slowly andaffects central vision by causing blurring and distortion. As itprogresses, the pulling of the membrane on the macula may causeswelling.

A calcineurin inhibitor of the present disclosure is preferably animmunophilin-binding compound having calcineurin inhibitory activity.Immunophilin-binding calcineurin inhibitors are compounds formingcalcineurin inhibiting complexes with immunophilins, e.g. cyclophilinand macrophilin. Examples of cyclophilin-binding calcineurin inhibitorsare cyclosporines or cyclosporine derivatives (hereinaftercyclosporines) and examples of macrophilin-binding calcineurininhibitors are ascomycin (FR 520) and ascomycin derivatives (hereinafterascomycins). A wide range of ascomycin derivatives are known, which areeither naturally occurring among fungal species or are obtainable bymanipulation of fermentation procedures or by chemical derivatization.Ascomycin-type macrolides include ascomycin, tacrolimus (FK506),sirolimus and pimecrolimus.

Cyclosporine, originally extracted from the soil fungus Potypaciadiuminfilatum, has a cyclic 11-amino acid structure and includes e.g.Cyclosporines A through I, such as Cyclosporine A, B, C, D and G.Cyclosporine binds to the cytosolic protein cyclophilin ofimmunocompetent lymphocytes, especially T-lymphocytes, forming acomplex. The complex inhibits calcineurin, which under normalcircumstances induces the transcription of interleukin-2 (IL-2).Cyclosporine also inhibits lymphokine production and interleukinrelease, leading to a reduced function of effector T-cells.

Voclosporin is a next-generation calcineurin inhibitor that is a morepotent and less toxic semi-synthetic derivative of cyclosporine A. Likeother molecules of this class, voclosporin reversibly inhibitsimmunocompetent lymphocytes, particularly T-lymphocytes, and alsoinhibits lymphokine production and release. This action is primarilymediated through inhibition of calcineurin, a phosphatase enzyme foundin the cytoplasm of cells. Voclosporin has a single carbon extensionwith double bond that has been shown to extend deeper into thelatch/regulatory region of calcineurin. In an embodiment, thecompositions of the present disclosure comprise the trans-version ofvoclosporin, trans-ISA247 CAS RN 368455-04-3 which is described in, forexample, US Patent Publication No.: 2006/0217309, which is herebyincorporated herein by reference. Further compositions of voclosporinare described, for example, in U.S. Pat. No. 7,060,672, which is herebyincorporated herein by reference.

Tacrolimus (FK506) is another calcineurin inhibitor which is also afungal product, but has a macrolide lactone structure. Tacrolimus hasbeen used as an immunosuppressant in conjunction with liver, kidney,heart, lung and heart/lung transplants. Tacrolimus has also been shownto inhibit the production of IL-2. Tacrolimus binds to an immunophilin(FK-binding protein 12, FKBP12), followed by binding of the complex tocalcineurin to inhibit its phosphatase activity.

Sirolimus (rapamycin) is a microbial product isolated from theactinomycete Streptomyces hygroscopicus. Sirolimus binds to animmunophilin (FK-binding protein 12, FKBP12) forming a complex, whichinhibits the mammalian target of rapamycin (mTOR) pathway throughdirectly binding the mTOR Complex1 (mTORC1). Sirolimus inhibits theresponse to interleukin-2 (IL-2) and thereby blocks activation of T- andB-cells. By contrast, tacrolimus and cyclosporine inhibit the productionof IL-2.

Pimecrolimus is a new calcineurin inhibitor which has been found to haveantifungal properties against Malassezia spp., as does tacrolimus.

Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin,tacrolimus, pimecrolimus, an analog thereof, or a pharmaceuticallyacceptable salt thereof, can be utilized in a mixed micellar compositionof the present disclosure. In an embodiment, the calcineurin inhibitoris voclosporin.

mTOR inhibitors such as sirolimus (rapamycin), temsirolimus, everolimus,an analog thereof, or a pharmaceutically acceptable salt thereof, can beutilized in a mixed micellar composition of the present disclosure.

The present disclosure provides pharmaceutical compositions that includea calcineurin inhibitor or an mTOR inhibitor, a first surfactant with anHLB index greater than about 10, and a second surfactant with an HLBindex of greater than about 13, wherein the pharmaceutical compositionforms mixed micelles. Typically, the mixed micelles are provided in anaqueous solution such that topical application of the compositions isachieved. In an embodiment, an absolute difference between the HLB indexof the first surfactant and the HLB index of the second surfactant isgreater than about 3. The compositions can be used in topicalapplication to the eye to treat a variety of ocular conditions,including both anterior segment and posterior segment conditions.

In an embodiment, a pharmaceutical composition of the present disclosurecomprises cyclosporine A, a first surfactant with an HLB index greaterthan about 10, and a second surfactant with an HLB index of greater thanabout 13. In an embodiment, the composition comprises cyclosporine A,vitamin E TPGS and octoxynol-40. In an embodiment, a mixed micellarcomposition of the present disclosure comprises voclosporin, a firstsurfactant with an HLB index greater than about 10, and a secondsurfactant with an HLB index of greater than about 13. In an embodiment,the composition comprises voclosporin, vitamin E TPGS and octoxynol-40.In an embodiment, a mixed micellar composition of the present disclosurecomprises tacrolimus, a first surfactant with an HLB index greater thanabout 10, and a second surfactant with an HLB index of greater thanabout 13. In an embodiment, the composition comprises tacrolimus,vitamin E TPGS and octoxynol-40. In an embodiment, a mixed micellarcomposition of the present disclosure comprises an mTOR inhibitor, afirst surfactant with an HLB index greater than about 10, and a secondsurfactant with an HLB index of greater than about 13. In an embodiment,the mTOR inhibitor is selected from one of sirolimus, temsirolimus,everolimus, an analog thereof, or a pharmaceutically acceptable saltthereof. In an embodiment, the composition comprised sirolimus, vitaminE TPGS and octoxynol-40. In another embodiment, a mixed micellarcomposition of the present disclosure comprises pimecrolimus, a firstsurfactant with an HLB index greater than about 10, and a secondsurfactant with an HLB index of greater than about 13. In an embodiment,the composition comprises pimecrolimus, vitamin E TPGS and octoxynol-40is disclosed.

In an embodiment of the present disclosure, two surfactants are used togenerate a mixed micellar formulation of voclosporin, resulting in anincrease in voclosporin's aqueous solubility and bioavailability. In anembodiment, the mixed micellar structure includes a first surfactantwith an HLB index greater than about 10, and a second surfactant with anHLB index of greater than about 13. In an embodiment, an absolutedifference between the HLB index of the first surfactant and the HLBindex of the second surfactant is greater than about 3.

In an embodiment, the first surfactant having an HLB greater than about10 is selected from various chemical derivatives of vitamin E with esterand ether linkages of various chemical moieties to polyethylene glycolof various lengths. Particularly preferred are vitamin E tocopherolpolyethylene glycol succinate (TPGS) derivatives with PEG molecularweights between about 500 and 6000 Da. In a preferred embodiment, thevitamin E polymeric derivative with an HLB index greater than about 10is vitamin E tocopherol polyethylene glycol 1000 succinate (Vitamin ETPGS, tocopherlosan). In an embodiment, the vitamin E TPGS is present inthe composition from about 0.01 wt % to about 20 wt %/volume. In anembodiment, the vitamin E TPGS is present in the composition from about0.1 wt % to about 10 wt %/volume. It should be understood thatthroughout the specification the term weight percent (wt %) refers tomass per unit volume, unless otherwise specified.

Vitamin E Tocopherol Polyethylene Glycol 1000 Succinate (Vitamin E TPGS)is an amphipathic excipient which is a water soluble derivative ofnatural-source vitamin E. Vitamin E TPGS, or PEGylated vitamin E, is avitamin E derivative in which polyethylene glycol subunits are attachedby a succinic acid diester at the ring hydroxyl of the vitamin Emolecule. Vitamin E TPGS is a hydrophilic non-ionic surfactant with anHLB index of about 13. Various chemical derivatives of vitamin E TPGSincluding ester and ether linkages of various chemical moieties areincluded within the definition of vitamin E TPGS. In addition to servingas a source of water-soluble vitamin E, vitamin E TPGS has beensuggested for use as an emulsifier, solubilizer, absorption enhancer,and a vehicle for lipid-soluble drug delivery formulations.

In an embodiment, the second surfactant has a HLB greater than 13 is ahydrophilic polyethylene glycol (PEG)-alkyl ether surfactant orpolyethylene glycol (PEG)-alkyl aryl ether surfactant. In an embodiment,this surfactant is selected from a PEG 5-100 octyl phenyl ether whichhas an HLB greater than about 13. The PEG octylphenyl compound isselected from the group consisting of octoxynol-9, octoxynol-10,octoxynol-11, octoxynol-12, octoxynol-13, octoxynol-16, octoxynol-20,octoxynol-25, octoxynol-30, octoxynol-33, octoxynol-40, andoctoxynol-70. In an embodiment, the PEG-alkyl phenyl ether surfactant isoctoxynol-40. In an embodiment, the surfactant with an HLB greater thanabout 10 is selected from a PEG-5-100 nonyl phenyl ether; tyloxapol(ethoxylated p-tert-octylphenol formaldehyde polymer), a PEG-fatty acidmonoester surfactant, a PEG-glycerol fatty acid ester, and aPEG-sorbiton fatty acid ester. PEG-Fatty acid monoester surfactantsinclude, but are not limited to, PEG-15 oleate, PEG-20 laurate, PEG-20oleate, PEG-20 stearate, PEG-32 laurate, PEG-32 oleate, PEG-32 stearate,PEG-40 laurate, PEG-40 oleate, and PEG-40 stearate. PEG-Glycerol fattyacid esters include, but are not limited to, PEG-15 glyceryl lauratePEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, and PEG-20 glyceryl stearate. PEG-sorbiton fatty acid estersinclude, but are not limited to, PEG-4 sorbiton monolaurate, PEG-4sorbiton monostearate, PEG-5 sorbiton monooleate, PEG-20 sorbitonmonolaurate, PEG-20 sorbiton monopalmitate, PEG-20 sorbitonmonostearate, and PEG-20 sorbiton monooleate. In an embodiment, thesecond surfactant with HLB greater than about 13 is octoxynol-40.Octoxynol-40 (IGEPAL CA-897) has an HLB index of about 18. In anembodiment, the octoxynol-40 is present in the composition from about0.001 wt % to about 10 wt %/volume. In an embodiment, the octoxynol-40is present in from about 0.01 wt % to about 5.0 wt %/volume.

Calcineurin inhibitors and mTOR inhibitors which can be formulatedaccording to the present disclosure include, but are not limited to,cyclosporine A, voclosporin (LX211), ascomycin, tacrolimus (FK506),sirolimus, everolimus, and pimecrolimus, including their analogs,pharmaceutically acceptable salts, esters, and prodrugs. Furthercontemplated are mixtures of a calcineurin or an mTOR inhibitor with oneor more drugs, vitamins, and diagnostic agents. A preservative may ormay not be used to preserve the formulations. In an embodiment, amixture of defined amounts of octoxynol-40 forms mixed micelles withvitamin E TPGS, creating stability and solubility for a water-insolubledrug that fills the inner core of the mixed micelle. In an embodiment,the mixed micellar composition comprises a calcineurin inhibitor,vitamin E TPGS and octoxynol-40. The mixed micellar formulation is aclear, homogenous aqueous solution of the calcineurin inhibitor or mTORinhibitor. In an embodiment, the Vitamin E TPGS contributes to thesolubilization of the calcineurin inhibitor or mTOR inhibitor and mayreduce ocular discomfort in aqueous conditions. In an embodiment, theoctoxynol-40 contributes to the reduction of ocular discomfort, and tothe formation of a stable, mixed micellar formulation that is opticallyclear.

In the compositions of the presently disclosed embodiments, thecalcineurin inhibitor or mTOR inhibitor is present at concentrationsranging from about 0.01 weight percent (wt %) to about 10 wt %, fromabout 0.1 to about 3.0 wt %. In an embodiment, the compositions of thepresent disclosure comprise voclosporin at about 0.2 to about 0.5 wt %,as illustrated in the examples. In an embodiment, the Vitamin E TPGSconcentration is from about 0.01 to about 20 wt %, from about 0.1 toabout 5 wt %. Octoxynol-40 or its homolog mixtures are present atconcentrations from about 0.001 to about 10 wt %, from about 0.01 toabout 3.0 wt %. In an embodiment, the total amount of surfactants in thecompositions of the present disclosure is 30 percent or less of thetotal composition with the remaining major component being water.

In an embodiment, a composition of the present disclosure comprisesabout 0.2 wt % of voclosporin, about 2.5 wt % of vitamin E TPGS, andabout 2.0 wt % octoxynol-40. In an embodiment. a composition of thepresent disclosure comprises about 0.5 wt % of voclosporin, about 3.5 wt% of vitamin E TPGS, and about 2.0 wt % octoxynol-40. In anotherembodiment, a composition of the present disclosure comprises about 2.0wt % voclosporin.

Site-specific delivery to the back-of-the-eye, including the choroid,and particularly the retina, is one of the challenges facing researchersin the field of therapeutic ophthalmology. There is growing but unmetneed for drug carriers that reach the retina at appropriate therapeuticlevels following topical administration. As will be shown in theExamples that follow, it has been found that after topicaladministration of a composition of the presently disclosed embodiments,the calcineurin inhibitor or mTOR inhibitor drug is able to reach theback of the eye, thus providing a treatment for back-of-the-eye ocularconditions.

The compositions of the present disclosure can be used as a topicallyapplied drug delivery platform for delivery of a variety of hydrophobic,water-insoluble drugs, such as a calcineurin inhibitor or mTOR inhibitorto the back-of-the-eye for various back-of-the-eye conditions. Suitableclasses of water-insoluble drugs include, but are not limited to,peptides, eicosanoids (e.g. prostacyclins and prostaglandins),anti-inflammatory drugs, autonomic drugs (e.g. beta-blockers,alpha-blockers, beta-agonists, and alpha-agonists), biologics, genetherapy agents (e.g. viral vectors), anti-infectives (e.g. antifungals,antibiotics, and antivirals), retinoids, RNAi, photo sensitizers,steroids (e.g., estrogens and derivatives thereof), mixture drugs,immuno-modulators, chemotherapeutic agents, G-coupled protein receptorantagonists, receptor tyrosine kinase (RTK) inhibitors, growth hormoneinhibitors, integrin inhibitors, Sdf1/CXCR4 pathway inhibitors, and nAChreceptor antagonists. Preferably, the water-insoluble drug is acalcineurin inhibitor or an mTOR inhibitor.

The compositions of the present disclosure can be used as a topicallyapplied drug delivery platform for delivery of a corticosteroid to theback-of-the-eye to treat, for example, DME. Examples of corticosteroidsinclude, but are not limited to, prednisolone, hydrocortisone,triamcinolone and budesonide.

The compositions of the present disclosure can be used as a topicallyapplied drug delivery platform for delivery of a non-steroidalanti-inflammatory drug (NSAID) to the back-of-the-eye to treat, forexample, DME. Examples of NSAIDs include, but are not limited to, Cox-2inhibitors such as celecoxib, ruboxistaurin and nimesulide.

The compositions of the present disclosure can be used as a topicallyapplied drug delivery platform for delivery of an anti-growth factormolecule to the back-of-the-eye to treat, for example, AMD. Examples ofanti-growth factor molecules include, but are not limited to, vascularendothelial growth factor (VEGF) inhibitors such as, pegaptanib(macugen), ranibizumab (lucentis), and bevacizumab (avastin).

In an embodiment, a mixed micellar composition of the present disclosurehaving either a calcineurin inhibitor or mTOR inhibitor that fills theinner core of the mixed micelle, can be used in topical application tothe eye in a method to treat a back-of-the-eye ocular condition. In anembodiment, calcineurin inhibitor or mTOR inhibitor is present in thecomposition at concentrations from about 0.01 weight percent (wt %) toabout 10 wt %, preferably from about 0.1 wt % to about 3.0 wt %. In anembodiment, the calcineurin inhibitor or mTOR inhibitor is voclosporin,and the voclosporin is present in the composition at a concentrationfrom about 0.2 wt % to about 0.5 wt %. In an embodiment, Vitamin E TPGSis present in the composition at concentrations from about 0.01 wt % toabout 20 wt %, preferably from about 0.1 wt % to about 5 wt %. In anembodiment, Octoxynol-40 or its homolog mixtures are present in thecomposition at concentrations from about 0.001 wt % to about 10 wt %,preferably from about 0.01 wt % to about 3.0 wt %. Preferably, the totalamount of surfactants in the compositions of the presently disclosedembodiments is about 30 percent or less of the total composition withthe remaining major component being water.

In an embodiment, a mixed micellar composition of the presentlydisclosed embodiments comprises about 0.2 wt % of voclosporin, about 2.5wt % of vitamin E TPGS, and about 2.0 wt % octoxynol-40. In anembodiment, a mixed micellar composition of the presently disclosedembodiments comprises about 0.5 wt % of voclosporin, about 3.5 wt % ofvitamin E TPGS, and about 2.0 wt % octoxynol-40. In another embodiment,a mixed micellar composition of the presently disclosed embodimentscomprises voclosporin at about 2.0 wt %.

At present, most ocular diseases are treated with the topicalapplication of solutions administered as eye drops for water-solubledrugs and as ointments or aqueous suspensions for water-insoluble drugs.These dosage forms account for approximately 90% of currently marketedformulations. The cornea represents a primary pathway for ocularpenetration of topically applied drugs. Drug absorption primarily takesplace through the cornea and into the aqueous humor and diffuses to theposterior segment. Drug can diffuse into the iris root and subsequentlyinto the posterior chamber aqueous humor and into the posterior tissues.Drug can enter directly through the pars plana without encountering theblood-retinal barrier. Drug can diffuse across the sclera by lateraldiffusion followed by penetration of Bruch's membrane and the retinalpigment epithelium (RPE). To a lesser extent, drug can be absorbed intothe systemic circulation either through the conjunctival vessels or vianasolacrimal duct and gain systemic access to the retinal vessels.

As shown in the Examples below, therapeutic levels of voclosporin werenoticed 24-hours post-administration of a pharmaceutical composition ofthe present disclosure, indicating that once daily (QD) dosing with theaqueous mixed micellar compositions of the presently disclosedembodiments is possible. As shown in the Examples, voclosporin, given inthe mixed micellar composition of the present disclosure, can bedetected at high levels in the choriod/retina, while low levels ofvoclosporin are detected in the vitreous humor. The calcineurininhibitor voclosporin is reaching the back of the eye when topicallyapplied in the mixed micellar formulations described herein.

The compositions of the present disclosure may also contain othercomponents such as, but not limited to, additives, adjuvants, buffers,tonicity agents, bioadhesive polymers, and preservatives. In any of thecompositions of this disclosure for topical to the eye, the mixtures arepreferably formulated at about pH 5 to about pH 8. This pH range may beachieved by the addition of buffers to the composition as described inthe examples. In an embodiment, the pH range in the composition in aformulation is about pH 6.6 to about pH 7.0. It should be appreciatedthat the compositions of the present disclosure may be buffered by anycommon buffer system such as phosphate, borate, acetate, citrate,carbonate and borate-polyol complexes, with the pH and osmolalityadjusted in accordance with well-known techniques to properphysiological values. The mixed micellar compositions of the presentdisclosure are stable in buffered aqueous solution. That is, there is noadverse interaction between the buffer and any other component thatwould cause the compositions to be unstable.

Tonicity agents include, for example, mannitol, sodium chloride,xylitol, etc. These tonicity agents may be used to adjust the osmolalityof the compositions. In one aspect, the osmolality of the formulation isadjusted to be in the range of about 250 to about 350 mOsmol/kg. In apreferred aspect, the osmolality of the formulation is adjusted tobetween about 280 to about 300 mOsmol/kg.

An additive such as a sugar, a glycerol, and other sugar alcohols, canbe included in the compositions of the present disclosure.Pharmaceutical additives can be added to increase the efficacy orpotency of other ingredients in the composition. For example, apharmaceutical additive can be added to a composition of the presentdisclosure to improve the stability of the calcineurin inhibitor or mTORinhibitor, to adjust the osmolality of the composition, to adjust theviscosity of the composition, or for another reason, such as effectingdrug delivery. Non-limiting examples of pharmaceutical additives of thepresent disclosure include sugars, such as, trehalose, mannose,D-galactose, and lactose. In an embodiment, the sugars can beincorporated into a composition prior to hydrating the thin film (i.e.,internally). In another embodiment, the sugars can be incorporated intoa composition during the hydration step (i.e., externally) (see Example17). In an embodiment, an aqueous, clear, mixed micellar solution of thepresent disclosure includes additives such as sugars.

In an embodiment, compositions of the present disclosure furthercomprise one or more bioadhesive polymers. Bioadhesion refers to theability of certain synthetic and biological macromolecules andhydrocolloids to adhere to biological tissues. Bioadhesion is a complexphenomenon, depending in part upon the properties of polymers,biological tissue, and the surrounding environment. Several factors havebeen found to contribute to a polymer's bioadhesive capacity: thepresence of functional groups able to form hydrogen bridges (—OH, COOH),the presence and strength of anionic charges, sufficient elasticity forthe polymeric chains to interpenetrate the mucous layer, and highmolecular weight. Bioadhesion systems have been used in dentistry,orthopedics, ophthalmology, and in surgical applications. However, therehas recently emerged significant interest in the use of bioadhesivematerials in other areas such as soft tissue-based artificialreplacements, and controlled release systems for local release ofbioactive agents. Such applications include systems for release of drugsin the buccal or nasal cavity, and for intestinal or rectaladministration.

In an embodiment, a composition of the present disclosure includes atleast one bioadhesive polymer. The bioadhesive polymer can enhance theviscosity of the composition and thereby increase residence time in theeye. Bioadhesive polymers of the present disclosure include, forexample, carboxylic polymers like Carbopol® (carbomers), Noveon®(polycarbophils), cellulose derivatives including alkyl and hydroxyalkylcellulose like methylcellulose, hydroxypropylcellulose,carboxymethylcellulose, gums like locust beam, xanthan, agarose, karaya,guar, and other polymers including but not limited to polyvinyl alcohol,polyvinyl pyrollidone, polyethylene glycol, Pluronic®(Poloxamers),tragacanth, and hyaluronic acid; phase-transition polymers for providingsustained and controlled delivery of enclosed medicaments to the eye(e.g., alginic acid, carrageenans (e.g., Eucheuma), xanthan and locustbean gum mixtures, pectins, cellulose acetate phthalate,alkylhydroxyalkyl cellulose and derivatives thereof, hydroxyalkylatedpolyacrylic acids and derivatives thereof, poloxamers and theirderivatives, etc. Physical characteristics in these polymers can bemediated by changes in environmental factors such as ionic strength, pH,or temperature alone or in combination with other factors. In anembodiment, the optional one or more bioadhesive polymers is present inthe composition from about 0.01 wt % to about 10 wt %/volume, preferablyfrom about 0.1 to about 5 wt %/volume. In an embodiment, thecompositions of the present disclosure further comprise at least onehydrophilic polymer excipient selected from, for example, PVP-K-30,PVP-K-90, HPMC, HEC, and polycarbophil. In an embodiment, the polymerexcipient is selected from PVP-K-90, PVP-K-30 or HPMC. In an embodiment,the polymer excipient is selected from PVP-K-90 or PVP-K-30.

In an embodiment, if a preservative is desired, the compositions mayoptionally be preserved with any well-known system such as benzylalcohol with/without EDTA, benzalkonium chloride, chlorhexidine,Cosmocil® CQ, or Dowicil® 200.

The ophthalmic compositions can be administered topically to the eye asbiocompatible, aqueous, clear mixed micellar solutions. The compositionshave the drugs incorporated and/or encapsulated in micelles which aredispersed in an aqueous medium.

In an embodiment, the present disclosure provides a method of preparinga mixed micelle composition that includes mixing a calcineurin or mTORinhibitor with a first surfactant having an HLB index greater than 10 ina solvent to form a solvent solution; evaporating the solvent solutionto form a near-solid matter; hydrating the near-solid matter with anaqueous solution comprising a second surfactant having an HLB indexgreater than 13 to form a mixture; and dissolving the mixture to producethe mixed micelle composition, where the resulting composition isoptically clear.

Suitable solvents that can be used in preparing the mixed micellecompositions of the present disclosure include short-chain alcohols, forexample, methanol, ethanol, n-propanol, isopropanol, and butanol, aswell as, chloroform, acetone, methylene chloride, dimethyl dulfoxide,dimethyl formamide and propylene glycol. The combination of two or threeshort chain alcohols may be used. Volatile organic solvents likechloroform and acetone may be used in combination with short chainalcohols. In an embodiment, the present disclosure provides a method ofpreparing a mixed micelle composition that includes mixing a calcineurininhibitor with vitamin E TPGS in a short-chain alcohol to form ashort-chain alcoholic solution; evaporating the short-chain alcoholicsolution to form a near-solid matter; hydrating the near-solid matterwith an aqueous solution comprising octoxynol-40 to form a mixture; anddissolving the mixture to produce the mixed micelle composition, wherethe resulting composition is optically clear.

In an embodiment, the short-chain alcohol is ethanol. In an embodiment,the present disclosure provides a method of preparing a mixed micellecomposition that includes mixing a calcineurin inhibitor with vitamin ETPGS and octoxynol-40 in ethanol to form an ethanolic solution. In anembodiment, the ethanol is 95% ethanol. In another embodiment, themethod provides for evaporating the ethanolic solution to form anear-solid matter. The near-solid matter may be resultant from rotaryvacuum evaporation of the ethanolic solution, in which case thenear-solid matter may be a thin film. The near-solid matter can also beresultant from evaporation of the ethanolic solution by, for example,lyophilization, freeze-drying, spray-drying, or by use of large andsmall scale evaporators, such as film evaporators, centrifugalevaporators, and vortex evaporators. The near-solid matter will beessentially free of ethanol (about <2% EtOH), but may contain up toabout 5% water. In an embodiment, the method provides for hydrating thenear-solid matter with an aqueous solution; and dissolving the mixtureto produce the mixed micelle composition, wherein the resultingcomposition is optically clear. The dissolving step may be performed bysonication, mixing, vortexing, stirring, mixing by rotary motion in arotary evaporator and/or shaking the near-solid matter in the aqueoussolution, or by other methods known in the art. In an embodiment, themethod further comprises mixing a bioadhesive polymer into the aqueoussolution prior to the hydrating step. In an embodiment, the bioadhesivepolymer is selected from PVP-K-30, PVP-K-90, HPMC, HEC, andpolycarbophil. In an embodiment, the bioadhesive polymer is selectedfrom PVP-K-30 or PVP-K-90. In an embodiment, the calcineurin inhibitorin the mixed micellar composition is voclosporin. In an embodiment, thevoclosporin is present from about 0.001% to about 10% in the mixedmicelle composition.

Pharmaceutically acceptable packaging materials for the compositionsinclude, but are not limited to, polypropylene, polystyrene, low densitypolyethylene (LDPE), high density polyethylene (HDPE), polycarbonate,polyvinylidine chloride, and other materials known to those skilled inthe art. The compositions can be packaged aseptically employingblow-fill-seal technology. Blow-fill-seal (BFS) describes an asepticfilling process in which hollow containers are blow molded, filled withsterile product, and sealed, all in one continuous machine cycle. Thetechnology is an alternative to conventional aseptic filling and cappingoperations, often providing cost savings through high output and processefficiency. In an embodiment, the compositions of the present disclosureare filled to single-use bottles, packets, vials, ampoules, LDPE BFScontainers, or HDPE BFS containers.

In an embodiment, multiple doses can be supplied as a plurality ofsingle-use packages. In another embodiment, the compositions areconveniently packaged in a bottle, container or device that allows formetered application, including containers equipped with a dropper fortopical ophthalmic application.

While the precise regimen is left to the discretion of the clinician, itis recommended that the compositions of the present disclosure betopically applied by placing one to two drops, or more, in each eye 1 to4 times daily. For example, the composition may be applied 1, 2, 3, 4,5, 6, 7 or 8 times a day, or more. In an embodiment, the composition aretopically applied by placing one to two drops in each eye once or twicedaily.

Artificial tears are lubricant eye drops used to treat, among otherthings, the dryness and irritation associated with deficient tearproduction in keratoconjunctivitis sicca (dry eyes). Artificial tearscan also be used to moisten contact lenses, as well as, moisten eyesduring an eye examination. Typically, artificial tears contain water,salts and polymers but lack the proteins found in natural tears. Variousartificial tears are available over-the-counter that contain ingredientssuch as carboxymethyl cellulose, hydroxypropyl methylcellulose (a.k.a.HPMC or hypromellose), and hydroxypropyl cellulose. Adverse effects havebeen shown in the known over-the-counter artificial tears, which areusually a consequence of the carboxymethyl cellulose component and othersimilar lubricants. These adverse effects include, for example, eyepain, irritation, continued redness, or vision changes.

In one aspect, unique biocompatible artificial tear compositions aredisclosed herein. The artificial tear compositions of the presentdisclosure are formulated as sterile, mixed micellar, aqueous solutionsthat include micelles formed from a first surfactant with an HLB indexgreater than about 10, and a second surfactant with an HLB index ofgreater than about 13. In an embodiment, the aqueous solution includesvarious ingredients chosen from one of hydrophilic polymer excipients,tonicity agents, buffers, preservatives, co-solvents or antioxidants.The biocompatible artificial tear compositions can be used to treatirritation, redness, swelling, allergic reaction, irritation due tocontact lens use, and corneal scratches and abrasions of the eyes.

Various hydrophilic polymer excipients may be employed including, butnot limited to, PVP-K-30, PVP-K-90, HPMC, HEC, and polycarbophil. In anembodiment, the hydrophilic polymer excipient is PVP-K-90.

Various tonicity agents may be employed to adjust the tonicity of theartificial tear compositions, preferably to that of natural tears. Forexample, sodium chloride, potassium chloride, magnesium chloride,calcium chloride and/or mannitol may be added to the compositions toapproximate physiological tonicity. In an embodiment, the tonicity agentis sodium chloride. Such an amount of tonicity agent will vary,depending on the particular agent to be added. In general, however, thecompositions will have a tonicity agent concentration of about 0.1-1.5%w/v.

An appropriate buffer system (e.g., sodium phosphate, sodium acetate,sodium citrate, sodium borate or boric acid in water) may be added toprevent pH drift under storage conditions. The particular concentrationwill vary, depending on the agent employed. In general, such aconcentration will range from about 0.02 to 2.0% w/v. In an embodiment,the buffer system includes sodium phosphate. Further, the sodiumphosphate may include both monosodium phosphate (i.e., monobasic) anddisodium phosphate (i.e., dibasic). In an embodiment, the pH of thebuffer system is adjusted such that an artificial tear composition ofthe presently disclosed embodiments ranges from about 6.5 to about 7.5.

Preservatives can be added to the artificial tear compositions of thepresent disclosure to increase the compositions shelf life and tofacilitate the use of multi-dose bottles. Examples of preservativesinclude, but are not limited to, Benzalkonium Chloride (BAC),Chlorobutanol, GenAqua (Sodium Perborate) and Polyquad(Polyquaternium-1).

A representative formulation for an artificial tear compositionaccording to the presently disclosed embodiments is shown in Example 16.Although specific concentration values are listed, those skilled in theart will recognize that the concentrations of the various ingredientscan be varied. Similarly, it may not be necessary to include all of theingredients listed in Example 16 in each artificial tear composition.

A method of preparing a mixed micelle composition includes mixing acalcineurin inhibitor or a mTOR inhibitor with a first surfactant havingan HLB index greater than about 10 and a second surfactant having an HLBindex of greater than about 13 in a solvent to form a solvent solution;evaporating the solvent solution to form a near-solid matter; hydratingthe near-solid matter with an aqueous solution; and dissolving thenear-solid mixture to produce the mixed micelle composition, wherein thecomposition is optically clear.

A method for treating an ocular disease in a patient in need thereofincludes administering topically to an eye of the patient a compositioncomprising a therapeutically effective amount of a calcineurin inhibitoror mTOR inhibitor, the composition further having vitamin E TPGS andoctoxynol-40, wherein the composition is an aqueous solution of mixedmicelles.

A method for treating, reducing, ameliorating, or alleviating aninflammatory ocular disease in an animal includes providing a mixedmicellar pharmaceutical composition having a calcineurin inhibitor or anmTOR inhibitor encapsulated in micelles, the micelles formed with afirst surfactant with an HLB index greater than about 10 and a secondsurfactant with an HLB index of greater than about 13; and administeringto the animal an amount of the pharmaceutical composition at a frequencysufficient to treat, reduce, ameliorate, or alleviate the inflammatoryocular disease.

A method for treating, reducing, ameliorating, or alleviating aback-of-the-eye condition or disorder in a subject includes providing amixed micellar pharmaceutical composition having a calcineurin inhibitorencapsulated in micelles formed with a first surfactant with an HLBindex greater than about 10 and a second surfactant with an HLB index ofgreater than about 13; and administering to the subject an amount of thepharmaceutical composition at a frequency sufficient to treat, reduce,ameliorate, or alleviate the back-of-the-eye condition or disorder.

EXAMPLES

In general, all reagents used are commercially available and usedwithout further purification unless indicated otherwise. Voclosporin(voclosporin, LX211, ISA247) was obtained from Isotechnika, Inc.,Edmonton, Alberta, Canada. The stock obtained from Isotechnika wasstored by Lux Biosciences at the New Jersey Center for Biomaterials;Cyclosporine A was obtained from Xenos Bioresources, Inc., SantaBarbara, Calif.; Sirolimus and Tacrolimus were obtained from HaoruiPharma-Chem, Inc. Vitamin E TPGS (NF Grade) was obtained from EastmanChemical Company, IGEPAL CA-897 (Octoxynol-40) was obtained from Rhodia,Inc., Distilled Deionized Water was prepared in house by use of EASYPure UV Compact Ultra Pure Water System, (Barnstead, Iowa). Kollidon® 30(PVP), and Kollidon® 90 F (Povidone K 90) were obtained from BASF.Hydroxyethyl Cellulose, 100 cps, and 5000 cps were obtained fromSpectrum, Methocel®, HPMC was obtained from Colorcon, Noveon®,Polycarbophil was obtained from Lubrizol Advanced Materials.

Example 1. General Preparation of a Basic Formulation

In order to make formulations at drug concentration of 0.02, 0.2, 0.4,0.5, and 1.0 wt %, the following protocols were employed. Drug basicformulations were made in the ratios shown in Table 1. In a firstprotocol, for example, calcineurin inhibitor and vitamin E TPGS requiredfor 50 mL were calculated, weighed, then mixed in 5 mL 95% ethanol,until a clear solution was obtained. The ethanolic solution wasevaporated under vacuum to get a thin film near-solid matter. Deionizedwater, 25 mL, was mixed with octoxynol-40 and the solution was added tothe thin film near-solid matter and sonicated for approximately 20 minto ensure complete formation of mixed micelles. The prepared 2×formulations were stored at room temperature. Alternatively, in a secondprotocol, amounts of drug, vitamin E TPGS and octoxynol-40 required for50 mL were calculated, weighed, then mixed in 5 mL 95% ethanol, andevaporated under vacuum to form a thin film near-solid matter. The thinfilm near-solid matter was then dissolved in 25 mL deionized water andsonicated or mixed by rotary motion in a rotary evaporator forapproximately 20 min to ensure complete formation of mixed micelles. Theprepared 2× formulations were stored at room temperature.

TABLE 1 Basic 2X Formulations (wt %/volume). Label/Ingredients 1 2 3Drug 0.4 0.8 1.0 Vitamin E TPGS 4.0 6.0 7.0 Octoxynol-40 1.0 1.0 1.0

Example 2. General Preparation of Formulations

Basic 2× Formulations shown in Table 1 were prepared as described in thesecond protocol described in Example 1. Basic formulations were preparedwhere the calcineurin or mTOR inhibitor was voclosporin, cyclosporine A,sirolimus and tacrolimus. In one preparation for 50 mL of formulation; abuffer mixture was prepared by dissolving amounts of components shown inTable 2 in 25 mL of deionized water to prepare a 2× buffer. The 2×buffer mixture was prepared both with and without added preservatives.

TABLE 2 Buffer Mixture. Amount Amount Amount for Amount for Componentsfor 50 mL for 50 mL 50 mL 50 mL Sodium Phosphate, 0.4048 g 0.4048 g0.4048 g 0.4048 g Dibasic Sodium Phosphate, 0.4645 g 0.4645 g 0.4645 g0.4645 g Monobasic EDTA    10 mg N.A.    10 mg N.A. Benzalkonium    10mg N.A. N.A.    10 mg chloride N.A. = not added

The required amount of polymer excipient shown in Table 3A was dispersedin 2.5 mL 2× buffer mixture and gently vortexed to get a clear solution.The basic 2× formulation was added in equal volume and mixed to getuniform solution. The pH of the solution was adjusted with NaOH or HClto a target of about 6.8. The osmolality of the solution was adjustedwith NaCl to be in the range of about 280-300 mOsmol/kg. The formulationwas sterilized by a nylon membrane filter (0.22 m) and then stored atroom temperature until use.

TABLE 3A Formulations. Label Ingredients 1 2 3 4 5 6 Basic Formulation(2X) 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL Buffer Mixture (2X) 2.5mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL PVP- K-30 (1.8%) 90 mg PVP-K-90 (1.2%) 60mg HPMC (0.5%) 25 mg HEC (0.5%) 25 mg Polycarbophil (0.5%) 25 mg Water2.5 mL Total Approx. Vol. 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL

In an alternative procedure for preparation of 100 mL of formulations,the basic 2× formulations shown in Table 1 were prepared usingvoclosporin. In order to make formulations at voclosporin concentrationsof 0.2, 0.4 and 0.5 wt %/volume, appropriate amounts of drug, vitamin ETPGS and octoxynol-40 required for 100 mL were calculated, weighed, thenmixed in 10 mL 95% ethanol, and evaporated under vacuum forapproximately 12 hours to form a thin film near-solid matter. The thinfilm near-solid matter was then dissolved in 50 mL deionized water andsonicated, or mixed by rotary motion in a rotary evaporator, forapproximately 20 minutes to ensure complete formation of mixed micelles;then stored at room temperature. The required amount of polymerexcipient shown in Tables 3B and 3C was dispersed in 40 mL deionizedwater and stirred to get a clear polymer solution. The other componentsshown in Tables 3B and 3C were added to the 50 mL basic 2× formulationand stirred well to get clear buffered solution. The clear bufferedsolution was slowly transferred into the clear polymer solution andmixed well. The pH of the solution was adjusted with NaOH or HCl to atarget of about 6.8. The osmolality of the solution was maintained inthe range of 280-300 mOsmol/kg. The volume was brought up to 100 mL withwater. The formulation was sterilized by a nylon membrane filter (0.22μm) and then stored at room temperature until use.

TABLE 3B Formulations. Label Ingredients 1 2 3 4 5 6 Basic Formulation(2X) 50 mL 50 mL 50 mL 50 mL 50 mL 50 mL Povidone-K-30 1.8 gPovidone-K-90 1.2 g Hydroxy propyl methyl 0.5 g cellulose Hydroxyethylcellulose 0.5 g Polycarbophil 0.9 g Sodium phosphate, dibasic 0.81 g0.81 g 0.81 g 0.81 g 0.81 g 0.81 g heptahydrate Sodium phosphate, 0.93 g0.93 g 0.93 g 0.93 g 0.93 g 0.93 g monobasic Sodium chloride 0.2 g 0.2 g0.2 g 0.2 g 0.2 g 0.2 g Water up to 100 mL 100 mL 100 mL 100 mL 100 mL100 mL

TABLE 3C Formulations. Label Ingredients 1 2 3 4 5 6 Basic Formulation50 mL 50 mL 50 mL 50 mL 50 mL 50 mL (2X) Povidone- K-30 1.8 gPovidone-K-90 1.2 g Hydroxy propyl 0.5 g methyl cellulose Hydroxyethyl0.5 g cellulose Polycarbophil 0.9 g Sodium phosphate, 0.81 g 0.81 g 0.81g 0.81 g 0.81 g 0.81 g dibasic heptahydrate Sodium phosphate, 0.93 g0.93 g 0.93 g 0.93 g 0.93 g 0.93 g monobasic Sodium chloride 0.2 g 0.2 g0.2 g 0.2 g 0.2 g 0.2 g Benzylkonium 0.02 g 0.02 g 0.02 g 0.02 g 0.02 g0.02 g chloride EDTA 0.02 g 0.02 g 0.02 g 0.02 g 0.02 g 0.02 g Water upto 100 mL 100 mL 100 mL 100 mL 100 mL 100 mL

One optimized formulation with voclosporin concentration at 0.2% wt%/vol. is shown in Table 3D.

TABLE 3D Formulation at 0.2 wt %/volume Voclosporin. Ingredient AmountVoclosporin (LX211) 0.2 g Vitamin E TPGS 2.0 g Octoxynol -40 2.0 gPVP-K-90 1.2 g Sodium Phosphate, Dibasic 0.81 g Sodium Phosphate,Monobasic 0.93 g Sodium Chloride 0.2 g Water up to 100 mL

Unless otherwise stated, data below are for formulations atapproximately 0.2% voclosporin. The viscosity of the formulation wasmeasured using cone and plate type viscometer. The clarity of theformulation was measured at 400 nm as described. Osmolality, pH,viscosity and absorbance at 400 nm for various formulations with 0.2%voclosporin are shown in Table 4A.

TABLE 4A Formulation Characteristics. Osmolality (mOsmol/kg) BeforeAfter addition addition Viscosity Absorbance Label/Ingredients of NaClof NaCl pH (Poise) at 400 nm Basic 010 — — 0.06 0.025 Formulation (1X)Basic 218 — 6.83 0.07 0.021 Formulation (2X) + Buffer Mixture (2X) B.For + BM + 248 347 6.85 0.07 0.032 PVP-K-30 B. For + BM + 224 303 6.810.08 0.034 PVP-K-90 B. For + BM + 228 311 6.82 0.11 0.025 HPMC B. For +BM + 237 283 6.80 0.08 0.031 HEC B. For + BM + 248 289 6.83 0.08 0.046Polycarbophil

A Cyclosporine A (CsA) formulation was prepared in the concentrationsshown in Table 4B a similar fashion as described in the second protocolin Example 1.

TABLE 4B CsA Formulation. Label/Ingredients wt %/vol Drug (CsA) 0.05Vitamin E TPGS 3 Octoxynol-40 0.02 Hydroxy Ethyl 0.2 CelluloseBenzalkonium Chloride 0.01 EDTA 0.01 Sodium Chloride 0.86 Water 100

The CsA formulation was adjusted to pH 6.88 and osmolality was 320mOsm/kg.

Example 3. Determination of Drug Content

Each formulation was analyzed for drug content by HPLC. The HPLC mobilephase consisted of acetonitrile/water/trifluoroacetic acid (75:25:0.1v/v/v) at a flow rate of 1 mL/min with elution of the compound ofinterest from a reversed-phase phenyl column (5 microns, 15×4.6 mm). Theabsorbance of the drug was measured at 210 nm with an UV detector andcompared with a standard curve of the target drug at various knownconcentrations. Observed peak for voclosporin eluted at approximately5.5 min.

Example 4. Filtration Efficiency Test

Various types of membranes were tested for use in filter sterilizationof formulations containing 0.2 wt % voclosporin. Membranes of 0.22 μMpore size were of various materials including nylon, teflon, andpolycarbonate. Recovery from membranes was evaluated by HPLCdetermination of drug content described above and compared tocentrifuged sample. Results for comparative filtration efficiency testsare shown in Tables 5A and 5B. Generally, nylon, teflon, andpolycarbonate membranes of 0.22 μM were each found acceptable for filtersterilization.

TABLE 5A Filtration Efficiency Test 1. Amount Drug Exp. of drug contentFormu- Conc. Conc. % in 50 (in lation Area (μg/mL) (μg/mL) RecoverymL(g) percent) Centrifuged Sample 1 4619728 2108.93 2200 95.86 0.1054460.211 2 4571834 2089.58 2200 94.98 0.104479 0.209 3 4589872 2096.87 220095.31 0.104843 0.210 Nylon Membrane 1 4537680 2075.78 2200 94.350.103789 0.208 2 4512464 2065.60 2200 93.89 0.10328 0.207 TeflonMembrane 1 4581475 2093.48 2200 95.16 0.104674 0.209 2 4567613 2087.882200 94.90 0.104394 0.209 3 4639411 2116.88 2200 96.22 0.105844 0.212

TABLE 5B Filtration Efficiency Test 2. Amount Drug Exp. of drug contentFormu- Conc. Conc % in 50 (in lation Area (μg/mL) (μg/mL) Recovery mL(g)percent) Centrifuged Sample 1 4531917 2073.45 2200 94.25 0.103673 0.2072 4506733 2063.28 2200 93.79 0.103164 0.206 3 4514394 2066.38 2200 93.930.103319 0.207 Polycarbonate Membrane 1 4491373 2057.08 2200 93.500.102854 0.206 2 4522797 2069.77 2200 94.08 0.103489 0.207 3 44829732053.68 2200 93.35 0.102684 0.205

Formulations at 0.2 wt % voclosporin with various bioadhesive polymerexcipients were prepared as described above in Table 3C. Formulationcharacteristics were measured and drug content was determined by HPLCafter filtration through a 0.22 μm nylon membrane. Results are shown inTable 6.

TABLE 6 Drug Content in 0.2 wt % Voclosporin Formulations. 1XBasicParameter Formulation PVP-K-30 PVP-K-90 HPMC HEC PC pH (beforeadjustment) 6.36 6.40 6.38 6.41 6.31 4.60 pH (after adjustment) 6.806.81 6.80 6.82 6.82 6.80 Osmolality (mOsm/kg) 325 328 303 280 297 330Viscosity (Poise) 0.11 0.12 0.13 0.17 0.16 0.19 Drug Content (%) 0.2030.202 0.192 0.191 0.173 0.183 by HPLC

Example 5. Clarity of the Formulations

The clarity of the formulations was measured visually and by recordingthe absorbance of the sample at 400 nm using an UV-visiblespectrophotometer. One milliliter of formulation and corresponding drugfree vehicles were placed in a plastic cuvette and absorbance wasrecorded at 400 nm. Water was used as blank. In a preferred aspect, themixed micellar formulation is a clear formulation with absorbance at 400nm of less than about 0.1. Absorbance at 400 nm is shown for variousformulations in Table 4A, and in dilution experiments in Tables 9-14.

Visual clarity was also used as a guideline in formulation trials. Forexample, Tables 7 and 8 show visual clarity at various wt % ofvoclosporin, vitamin E TPGS and octoxynol-40 in various 1× basicformulations, prepared as described in the second protocol in Example 1.

TABLE 7 Formulation Trials. Label/ Ingredients 1 2 3 4 Drug(Voclosporin) (wt %) 2.0 2.0 2.0 2.0 Vitamin E TPGS (food grade) (wt %)4.5 5.0 5.5 6.0 Octoxynol-40 (wt %) 3.0 3.0 3.0 3.0 Water up to (mL) 100100 100 100 Visual clarity milky milky milky milky

In Table 7, food grade vitamin E TPGS was used at the concentrationsshown; all samples were milky. In Table 8, samples 1 and 2 were visuallyclear, but samples 3 and 4 contained undissolved drug.

TABLE 8 Formulation Trials. Label/ Ingredients 1 2 3 4 Drug(Voclosporin) (wt %) 0.75 1.0 1.5 2.0 Vitamin E TPGS (wt %) 6.0 6.0 6.06.0 Octoxynol-40 (wt %) 4.0 4.0 4.0 4.0 Water up to (mL) 100 100 100 100Visual clarity clear clear cloudy cloudy

Example 6. Dilution Study of Voclosporin Formulations in ArtificialTears

Voclosporin formulations were evaluated in dilution studies. The goalwas to subject formulations to dilution under conditions similar to theeye. The voclosporin concentration was 0.2 wt % in each formulationtested. The formulations as described in Table 3A were each mixed 1:1,1:5 and 1:10 with various brands of artificial tears available over thecounter (OTC) in the pharmacy. Systane® (Lubricant Eye Drops, Alcon,Inc.; Visine® (Lubricant Eye Drops, Pfizer, Inc.; Refresh Tears®(Lubricant Eye Drops), Allergan, Inc.; and Hypo Tears® (Lubricant EyeDrops), Novartis, were employed. The measurements were taken underambient conditions. The data (absorbance at 400 nm) are shown in Tables9 to 14A. Results showed no increase in turbidity and hence noprecipitation of voclosporin out of solution.

TABLE 9 Sample Absorbance at 400 nm, pre-dilution. Absorbance Sample No.Formulations (400 nm) 1 PVP-K-30 0.020 2 PVP-K-90 0.018 3 HPMC 0.021 4HEC 0.019 5 Polycarbophil 0.192 6 Water 0.000 Tears Fluid Absorbance(400 nm) 7 Refresh Tears 0.000 8 Visine Tears 0.017 9 Systane Tears0.023 10 Hypo Tears 0.002

TABLE 10 Sample Absorbance at 400 nm, post-dilution. Sample Type of tearAbsorbance No: Formulation fluid Dilution factor (400 nm) 11 PVP-K-30Refresh Tears 2X 0.020 12 5X 0.014 13 10X  0.002 14 Visine Tears 2X0.011 15 5X 0.005 16 10X  0.002 17 Systane Tears 2X 0.019 18 5X 0.019 1910X  0.021 20 Hypo Tears 2X 0.013 21 5X 0.005 22 10X  0.041

TABLE 11 Sample Absorbance at 400 nm, post-dilution. Sample Type of tearAbsorbance No: Formulation fluid Dilution factor (400 nm) 23 PVP-K-90Refresh Tears 2X 0.012 24 5X 0.007 25 10X  0.004 26 Visine Tears 2X0.013 27 5X 0.006 28 10X  0.003 29 Systane Tears 2X 0.020 30 5X 0.020 3110X  0.031 32 Hypo Tears 2X 0.010 33 5X 0.005 34 10X  0.003

TABLE 12 Sample Absorbance at 400 nm, post-dilution. Sample Type of tearAbsorbance No: Formulation fluid Dilution factor (400 nm) 35 HPMCRefresh Tears 2X 0.010 36 5X 0.004 37 10X  0.001 38 Visine Tears 2X0.009 39 5X 0.005 40 10X  −0.001 41 Systane Tears 2X 0.018 42 5X 0.02143 10X  0.021 44 Hypo Tears 2X 0.009 45 5X 0.004 46 10X  0.002

TABLE 13 Sample Absorbance at 400 nm, post-dilution. Sample Type of tearAbsorbance No: Formulation fluid Dilution factor (400 nm) 47 HEC RefreshTears 2X 0.009 48 5X 0.004 49 10X  0.002 50 Visine Tears 2X 0.010 51 5X0.004 52 10X  0.002 53 Systane Tears 2X 0.020 54 5X 0.020 55 10X  0.02056 Hypo Tears 2X 0.010 57 5X 0.004 58 10X  0.003

TABLE 14A Sample Absorbance at 400 nm, post-dilution. Sample Type oftear Absorbance No: Formulation fluid Dilution factor (400 nm) 59Polycarbophil Refresh Tears 2X 0.052 60 5X 0.078 61 10X  0.054 62 VisineTears 2X 0.046 63 5X 0.086 64 10X  0.065 65 Systane Tears 2X 0.038 66 5X0.053 67 10X  0.047 68 Hypo Tears 2X 0.030 69 5X 0.013 70 10X  0.008

Further dilution studies were performed on the formulation shown inTable 3B, column 1, with buffered saline as diluent. Diluted formulationwas characterized and data are shown in Table 14B. Micellar stabilitywas confirmed to at least 20 fold dilution in buffered saline.

TABLE 14B Micellar Stability Upon Dilution. Dilution Osmolality ParticleSize DST RS Formulation Factor Appearance pH (mOsm/kg) (nm) PD (° C.)Time No polymer 0X Clear 6.78 326 10.6 0.037 55 3 min 4X Clear 6.87 34012.2 0.161 60 3 min 40 sec 20X  Clear 7.08 300 20.8 0.264 65 2 min 100X Clear 7.25 301 339.6 0.537 — — DST—Dissociation Temperature,RS—Restabilization, PD—Polydispersity

Example 7. Dissociation Temperature for the Drug Free Formulations andFormulations Containing Voclosporin

Formulations shown in Table 3A were tested to determine dissociationtemperature with and without 0.2 wt % voclosporin/volume. A water bathat a constant temperature of ˜60° C. was prepared and used for testingof samples with drug. A glass vial containing the formulation wasinserted into the water bath with a thermometer inserted in theformulation. As soon as some turbidity was visually observed, atemperature reading was taken. The turbid solutions were cooled to roomtemperature and the drug went back into the mixed micelles with theresult that all solutions became clear again. The time forre-stabilization (reestablishment of visual clarity) was recorded. Datafor samples with voclosporin are shown in Table 15. A heat block wasused to heat and test samples without drug in a similar fashion. Datafor samples without voclosporin are shown in Table 16.

The data shows that in the absence of voclosporin, the dissociationtemperature of the micellar formulations generally is about 20-40degrees celsius higher than the dissociation temperature of the micellarformulation in the presence of voclosporin (with the exception of theHPMC-containing formulation). The decrease in the dissociationtemperature of the drug-containing micellar formulations indicates thatthe drug is incorporated into the micelles, and thereby solubilized.

TABLE 15 Dissociation Temperature of Formulation with 0.2 wt %Voclosporin. Time Required Sample Formulations with Temperature for No:0.2% Voclosporin. (° C.) Restabilization 1 Formulation without 44 6 minpolymer (basic) 2 PVP-K-30 46 5 min 30 sec 3 PVP-K-90 45 4 min 30 sec 4HPMC 44 2 min 5 HEC 43 5 min 6 Polycarbophil 43 ND ND = Not Determined

TABLE 16 Dissociation Temperature of Formulation without Voclosporin.Sample Temperature No: Drug free Formulations (° C.) 7 PVP-K-30 92 8PVP-K-90 90 9 HPMC 46 10 HEC 90 11 Polycarbophil 75

An additional thermal dissociation experiment was performed whereinvials containing 1 mL of 0.2% voclosporin formulations (basic, HPMC, andPVP-K-90) were heated in a water bath maintained at ˜50° C. for about 5minutes. The mixed micelles were destabilized and the solution becameturbid or milky white. The solutions were cooled to room temperature andthe drug went back into the mixed micelles with the result that allsolutions became clear again. The time for re-stabilization wasrecorded. The PVP-K-90 sample was recycled a second time with the sameresults.

Generally, formulations with an increased wt % of octoxynol-40 exhibitedan increase in the dissociation temperature and decreased theregeneration time (the time required for re-stabilization), as shown inTables 17 and 18.

TABLE 17 Dissociation Temperatures in Basic Formulations with 0.2 wt %Voclosporin and various wt % Octoxynol-40. Dissociation Time requiredSample Concentration of Temperature for re- No: Octoxynol-40 (° C.)stabilization 1 0.5% 46 7 min 30 sec 2 1.0% 53 6 min 10 sec 3 1.5% 55 5min 30 sec 4 2.0% 55 3 min 20 sec 5 2.5% 56 3 min

TABLE 18 Dissociation Temperatures in Basic Formulations with 0.5 wt %Voclosporin and various wt % Octoxynol-40. Dissociation Time requiredSample Concentration of Temperature for re- No: Octoxynol-40 (° C.)stabilization 1 0.5% 46 Not stabilized 2 1.0% 46 6 min 3 1.5% 47 5 min 42.0% 48 7 min 5 2.5% 49 7 min 30 sec 6 3.0% 49 7 min 30 sec

Another dissociation temperature experiment where the concentration ofoctoxynol-40 was increased from 0.5% to 2.5% in the PVP-K-90 formulationwith 0.2 wt % voclosporin resulted in an increase in dissociationtemperature from 45° C. to 55° C. The formulation reestablished to aclear solution within 3 minutes after cooling.

Addition of further excipients was evaluated to determine the effect ondissociation temperature. Addition of 5% PEG 400 to formulations asprepared in Table 3B with 0.2 wt % voclosporin resulted in similardissociation temperatures and slightly increased time required forre-stabilization, as shown in Table 19.

TABLE 19 Dissociation Temperature: Effect of Addition of 5% (v/v) PEG400. Sample Formulations with Dissociation Time Required for No.Voclosporin Temperature (° C.) re-stabilization 1 Formulation without 426 min polymer + 5% PEG 400 2 PVP-K-90 + 44 6 min 5% PEG 400 3 HPMC + 422 min 45 sec 5% PEG 400 4 HEC + 39 6 min 5% PEG 400

Addition of 1% HPMC to formulations as prepared in Table 3B with 0.2 wt% voclosporin and PVP-K-90 resulted in similar dissociationtemperatures, but a decreased time required for re-stabilization, asshown in Table 20.

TABLE 20 Dissociation Temperature: Effect of Addition of 1% HPMC toPVP-K-90 Formulation. Sample Formulation with Dissociation Time Requiredfor No. Voclosporin Temperature (° C.) re-stabilization 1 PVP-K-90 +HPMC 43 3 min 45 sec

Example 8. Particle Size Measurements

The mean particle size and polydispersity index of the mixed micellesare measured using dynamic light scattering technique (Brookhaven 90Plusparticle size analyzer, Holtsville, N.Y.), taking the average of threemeasurements. The different solutions were placed in disposable plasticcells. A sample volume of 200 μL was used for determining the particlesize. Particle size and polydispersity for formulations as prepared inExample 2 with 0.2 wt % voclosporin are shown in Table 21. Theformulation with 0.2 wt % voclosporin and PVP-K-90 exhibited an averagemicelle diameter of 13.3 nm with a very narrow size distribution and apolydispersity of 0.005. In contrast, the formulation with 0.2 wt %voclosporin and HEC exhibited an average micelle diameter of 23.8, but abroad, bimodal particle size distribution resulted in a largepolydispersity of 0.482.

TABLE 21 Particle Size Analysis. Formulation with Diameter Sample No.0.2 wt % Voclosporin (nm) Polydispersity 1 Formulation without 8.0 0.657polymer 2 PVP-K-30 19.8 0.206 3 PVP-K-90 13.3 0.005 4 HPMC 32.9 0.317 5HEC 23.8 0.482

Particle size, polydispersity, dissociation temperature andre-stabilization time for formulations with 0.2 wt % and 0.5 wt %voclosporin in formulations with 2% octoxynol-40 are shown in Tables 22and 23.

TABLE 22 Characteristics of Formulations Containing 0.2 wt % Voclosporinwith 2% Octoxynol-40. Dissociation Time Sample Osmolality Particle SizePolydispersity Temperature required for re- No Formulations (mOsm/kg)(nm) index (° C.) stabilization 1 Formulation 75 9.9 0.103 57 2 minwithout Buffer & polymer 2 Formulation 231 11.1 0.157 58 2 min 40 secwithout polymer 3 Formulation 248 10.5 0.083 65 3 min containing 3% OC-40 without polymer 4 PVP-K-30 (1.8%) 256 11.6 0.147 58 3 min 5 PVP-K-90(1.2%) 266 12.5 0.156 59 3 min 20 sec 6 HPMC (0.3%) 275 97.3 0.160 53 3min 7 HEC (0.3%) 233 83.9 0.166 59 2 min 50 sec

TABLE 23 Characteristics of Formulations Containing 0.5 wt % Voclosporinwith 2% Octoxynol-40. Dissociation Time Sample Osmolality Particle SizePolydispersity Temperature required for re- No Formulations (mOsm/kg)(nm) index (° C.) stabilization 1 Formulation 178 9.6 0.030 49 14 minwithout Buffer & polymer 2 Formulation 275 10.6 0.055 46 12 min withoutpolymer 3 Formulation 358 11.0 0.115 44 13 min containing 3%Octoxynol-40 without polymer 4 PVP-K-30 (1.8%) 284 12.7 0.189 47 12 min5 PVP-K-90 (1.2%) 281 21.8 0.251 48 12 min 50 sec

Example 9. Determination of Drop Weight and Volume

In order to determine the amount of calcineurin inhibitor delivered perdrop, the drop weight and volume was determined for each formulation.Since the drop size is dependent on the surface tension of theformulation, two formulations, as described in Table 3A, containing 0.2wt % voclosporin/volume were tested for delivered drop size and volume.The formulation containing PVP-K-90, and the formulation containingHPMC, 0.5 mL each, were filled individually into 0.8 mL capacity BFS(blow-fill-seal) containers provided by a manufacturing vendor. Thebottle material was LDPE and the study was conducted under ambientconditions. Ten drops of each formulation was squeezed into a tared dishand weighed. Similarly ten drops of formulations were squeezed in to themeasuring cylinder and volume was recorded. Data is shown in Tables 24and 25.

TABLE 24 Weight of 10 Drops. Weight of 10 drops (g) Sample No PVP-K-90HPMC 1 0.2843 0.2851 2 0.2829 0.2843 3 0.2838 0.2848 Average 0.28360.2847

TABLE 25 Volume of 10 Drops. Volume of 10 drops (mL) Sample No PVP-K-90HPMC 1 0.29 0.30 2 0.28 0.29 3 0.28 0.29 Average 0.283 0.293

Example 10. Stability Studies

Stability and formulation compatibility studies were performed in threetypes of bottles suitable for pharmaceutical delivery. Known volumes ofthe six formulations of Example 1 were transferred to three differenttypes of containers i.e., LDPE, polypropylene and polyvinylchloride andstored at room temperature. At predetermined time intervals (0, 6, 24and 48 hr) the samples were withdrawn from the containers and analyzedfor the drug content by HPLC method. None of the formulations stored invarious types of containers exhibited a decrease in drug content duringthe study period.

Example 11. Local Tolerability in Rabbits of Formulations Comprising aCalcineurin Inhibitor

A study was conducted in rabbits to test the tolerance of mixed micellarformulations containing voclosporin (1× basic formulation, Table 3A,column 1, at either 0.2 wt % or 0.5 wt % voclosporin, one rabbit each)against saline solution. Healthy young adult New Zealand albino rabbits(3-4 Kg) were used for the study. One drop (approximately 30 μL) ofsaline was placed in one eye and a drop of formulation with voclosporinwas placed in the other eye of the rabbit. No difference was noticed inthe following observed parameters: blinking of the eye, lacrimation,pupil size, redness, movement of the eye.

Example 12. Local Tolerability in Rabbits of Formulations Comprising aCalcineurin Inhibitor

Further studies were conducted in rabbits to test the tolerance ofvarious mixed micellar formulations. Formulations F1-F16 as shown inTables 26 and 27 were used for these studies.

TABLE 26 Formulations F1 to F8. Code F1 F2 F3 F4 F5 F6 F7 F8 Voclo-0.2%  0.2%  0.2% 0.2% 0.2% 0.2% 0.2% 0.2% sporin Vitamin E 2% 2% 3.5%3.5%  2%  2% 3.5% 3.5% TPGS OX-40 2% 3%  2%  3%  2%  3%  2%  3% PVP-K-90— — — — 0.6% 0.6% 0.6% 0.6%

TABLE 27 Formulations F9 to F16. Code F9 F10 F11 F13 F14 F14 F15 F16Voclosporin 0.2% 0.2% 0.2% 0.2% 0.02%   0.02%   0.02%  0.02%  Vitamin ETPGS  2%  2% 3.5% 3.5% 2% 2% 3.5% 3.5% OX-40  2%  3%  2%  3% 2% 3%  2% 3% PVP-K-90 1.2% 1.2% 1.2% 1.2% 1.2%  1.2%  1.2% 1.2%

Healthy young adult New Zealand albino rabbits (3-4 Kg) were used forthe study. One drop (approximately 30 μL) of a formulation withvoclosporin (LX211) was placed in an eye of the rabbit. Each formulationwas tested in triplicate.

Both eyes of each animal were examined by a board-certified veterinaryophthalmologist using a hand-held slit lamp and indirect ophthalmoscope.Both control and test eyes were graded according to conjunctivalcongestion, swelling, and discharge, aqueous flare, iris light reflexand involvement, corneal cloudiness severity and area, pannus,fluorescein examination and lens opacity using the Hackett/McDonaldscoring system (see, for example, Hackett, R. B. and McDonald, T. O.Ophthalmic Toxicology and Assessing Ocular Irritation. Dermatoxicology,5^(th) Edition. Ed. F. N. Marzulli and H. I. Maibach. Washington, D.C.:Hemisphere Publishing Corporation. 1996; 299-305 and 557-566.). In thefluorescein examination, approximately one drop of 0.9% sodium chloride,USP, was applied to the end of a fluorescein impregnated strip and thenapplied to the superior sclera of the left and right eyes (onefluorescein impregnated strip is used for each animal). After anapproximate 15 second exposure, the fluorescein dye was gently rinsedfrom each eye with 0.9% sodium chloride, USP. The eyes were thenexamined using a slit lamp with a cobalt blue filtered light source. Forthe lenticular examination approximately one drop of a short-actingmydriatic solution was instilled onto each eye in order to dilate thepupil. After acceptable dilation has occurred, the lens of each eye wasexamined using a slit-lamp biomicroscope.

The crystalline lens is readily observed with the aid of the slit-lampbiomicroscope, and the location of lenticular opacity can readily bediscerned by direct and retro illumination. The location of lenticularopacities can be arbitrarily divided into the following lenticularregions beginning with the anterior capsule: Anterior subcapsular,Anterior cortical Nuclear Posterior cortical, Posterior subcapsular,Posterior capsular. The lens is evaluated routinely during ocularevaluations and graded as either 0 (normal) or 1 (abnormal). Thepresence of lenticular opacities should be described and the locationnoted. Results for various formulations are shown in Tables 28 to 31.

TABLE 28 Tolerability Test Results in Rabbit Eyes for VariousFormulations at 0.2 wt % Voclosporin. Pre Treatment 1 Hour 24 Hour 72Hour Rabbit # F1 F2 F3 F4 F1 F2 F3 F4 F1 F2 F3 F4 F1 F2 F3 F4 I59 0% 0 01 1 0 0 0 0 I60 PVP- 0 0 0 0 0 0 0 0 I61 K-90 0 0 1 0 0 0 0 1 I62 1 1 11 1 2 2 0 I63 0 0 0 0 0 0 0 0 I64 0 0 0 1 0 0 0 0

TABLE 29 Tolerability Test Results in Rabbit Eyes for VariousFormulations at 0.2 wt % Voclosporin. Pre Treatment 1 Hour 24 Hour 72Hour Rabbit # F5 F6 F7 F8 F5 F6 F7 F8 F5 F6 F7 F8 F5 F6 F7 F8 I65 0.6% 00 0 0 0 0 0 0 I66 PVP- 1 1 1 1 0 0 0 0 I67 K-90 0 0 1 2 0 0 0 0 I68 0 00 0 1 0 0 0 I69 0 0 1 1 1 1 1 0 I70 0 0 1 1 0 0 0 0

TABLE 30 Tolerability Test Results in Rabbit Eyes for VariousFormulations at 0.2 wt % Voclosporin. Pre Treatment 1 Hour 24 Hour 72Hour Rabbit # F9 F10 F11 F12 F9 F10 F11 F12 F9 F10 F11 F12 F9 F10 F11F12 I71 1.2% 0 0 0 0 0 0 0 0 I72 PVP- 0 0 1 0 0 0 0 0 I73 K-90 0 0 0 0 00 0 0 I74 0 0 0 0 0 0 0 0 I75 0 0 0 0 0 0 0 0 I76 0 0 0 1 0 0 0 0

TABLE 31 Tolerability Test Results in Rabbit Eyes for VariousFormulations at 0.02 wt % Voclosporin Pre Treatment 1 Hour 24 Hour 72Hour Rabbit # F13 F14 F15 F16 F13 F14 F15 F16 F13 F14 F15 F16 F13 F14F15 F16 I77 1.2% 0 0 0 1 0 0 0 0 I78 PVP- 0 0 2 0 0 0 0 0 I79 K-90 0 0 00 0 0 0 0 I80 0 0 0 0 0 1 0 0 I35 0 0 0 0 0 0 0 0 I36 0 0 0 1 1 1 0 2

Example 13. Topical Voclosporin Clinical Study in Dogs with KCS

An open label, single group, pilot efficacy study evaluating topicalvoclosporin was designed and conducted. The study was intended todocument the efficacy of 0.2 wt % voclosporin in a composition accordingto the presently disclosed embodiments for the treatment of caninekeratoconjunctivitis sicca (KCS). The study covered assessment of tearproduction (as measured by the Schirmer Tear Test (STT)), the responseof clinical observation of the cornea, and participatingophthalmologists' overall assessment of efficacy.

Dogs diagnosed with chronic (>3 months in duration) immune-mediated KCSwere selected from the clinic populations of the North Carolina StateVeterinary Teaching Hospital. Diagnosis of immune-mediated KCS was madeby exclusion of other causes of KCS. Dogs selected to be entered intothis study had demonstration of residual lacrimal function and haveshown response to commercially available topical cyclosporine.

In this study, there was no washout period and animals were switcheddirectly from topical cyclosporine A (0.2% cyclosporine in petrolatum,USP; corn oil, NF; and Amerchol® CAB base (Optimmune® Schering PloughAnimal Health)) to 0.2 wt % voclosporin in a mixed micellar compositionaccording to the presently disclosed embodiments, given topically every12 hours. Physical and ophthalmic examinations were performed at 0, 7,14, and 28 days. The study was designed such that a favorable responseto the voclosporin would be considered a maintenance or increase of STTvalue compared to pre-study values.

Six dogs were entered and completed the study. For these 6 dogs, themean STT at day 0 was 21.9±SD 3.2 mm/min; at 7 days of therapy STT was22.4±4.0 mm/min; at 14 days STT was 20.3±2.5 mm/min, and at 30 days STTwas 21.0±1.9 mm/min. This clearly indicates that voclosporin hasmaintained the STT in these dogs for 30 days. See mean STT values inFIG. 1. All dogs have been comfortable without any signs of side effectsor irritation associated with the medication. No adverse effects werenoted in any animal during the 30 days treatment period.

Example 14. Robustness and Stability of Formulations

The robustness of a formulation according to the present disclosurecontaining 0.2 wt % voclosporin was tested by subjecting the samples tomultiple heat/cool cycles, refrigeration cycles, vigorous shaking orextended exposure to the sun light.

Thermal Cycling:

A set of glass vials containing formulation were placed in a water bathwith temperature set at −70° C. The samples were heated until thecloudiness appeared and then were cooled at room temperature for thesolution to become clear, which constituted one round of thermalcycling. The thermal cycling was repeated 5 or 10 times. Aftercompletion of the 5 or 10 thermal cycles, the samples were analyzed fordissociation temperature followed by regeneration time and micellar sizedetermination as described above.

Refrigeration Cycling:

A set of samples were subjected to the refrigerated conditions. Thesamples were placed in a refrigerator (4° C.) for 12 hours and thenbrought to room temperature and maintained at room temperature for 12hours. The thermal cycling was repeated 5 or 10 times. After completionof the 5 or 10 cycles, samples were analyzed for dissociationtemperature, followed by regeneration time and micellar sizedetermination as described above.

Vigorous Shaking:

Samples were placed on shaking platform and the shaker was operated at−75 rpm at room temperature. Samples were withdrawn after 4 hours or 24hours and analyzed for dissociation temperature, regeneration time andmicellar size as described above.

Sunlight Exposure:

Solutions were placed under direct sunlight for 4 hours. Post exposure,the sample were analyzed for dissociation temperature, followed by theregeneration time and micellar size as described above.

The mixed micellar formulation according to the present disclosurecontaining 0.2 wt % voclosporin was subjected to various stressconditions (heat/cool cycles, refrigeration/ambient cycles, vigorousshaking and exposure to the sun light). The mixed micellar compositionaccording to the presently disclosed embodiments containing 0.2 wt %voclosporin did not exhibit changes in the dissociation temperature,regeneration time and micelle size as shown in Table 32.

TABLE 32 Effect of stress on dissociation temperature, regeneration timeand micelle size, average of three replicate samples. DissociationRegeneration Micellar temperature time size No Description of Test (°C.) (min) (nm) PDI 1 Samples before subjected to 54.0 ± 1.0 2.5 13.3 ±0.2 0.193 ± 0.004 any stress 2 Samples subjected to 5 57.3 ± 0.6 3.016.0 ± 1.6 0.198 ± 0.020 cycles of heat/cool 3 Samples subjected to 1057.0 ± 1.0 3.0 15.9 ± 1.4 0.211 ± 0.10  cycles of heat/cool 4 Samplessubjected to 5 55.0 ± 1.0 2.6 ± 0.3 13.6 ± 0.4 0.195 ± 0.011refrigeration/ambient cycles 5 Samples subjected to 10 55.0 ± 1.7 3.013.3 ± 0.8 0.189 ± 0.10  refrigeration/ambient cycles 6 Samplessubjected to 4 54.6 ± 0.6 3.0 ± 0.1 14.2 ± 0.7 0.193 ± 0.008 hours ofshaking on a shaking platform 7 Samples subjected to 24 54.6 ± 0.6 2.8 ±0.3 14.1 ± 0.4 0.193 ± 0.003 hours of shaking on a shaking platform 8Samples subjected to 4 54.6 ± 0.6 2.8 ± 0.3 13.7 ± 0.4 0.194 ± 0.005hours of sun lightStability Study:

Solutions in triplicate were transferred into clean glass vials andstored at different temperatures (45° C., 30° C., RT and 4° C.). Atpredetermined time intervals (0, 7, 14 and 30 days) samples werewithdrawn and assessed for change in color, phase separation, pH, drugcontent, dissociation temperature, regeneration time and micellar size.

Samples stored at 30° C., RT and 4° C. for up to 30 days did not showchanges in color, phase, pH, drug content, dissociation temperature,regeneration time and micellar size. Solutions stored at 45° C. formedprecipitates indicating thermal instability of the formulation at hightemperatures.

Example 15. Preparation and Micellar Characterization of FormulationsContaining Various Calcineurin or mTOR Inhibitors

TABLE 33 Formulations containing various calcineurin and mTORinhibitors. Ingredient Amount for 100 mL Cyclosporine A 0.2 g — —Sirolimus — 0.2 g — Tacrolimus — — 0.2 g Vitamin E TPGS 2.5 g 2.5 g 2.5g Octoxynol-40 2.0 g 2.0 g 2.0 g PVP-K-90 1.2 g 1.2 g 1.2 g SodiumPhosphate, Dibasic 0.81 g 0.81 g 0.81 g Sodium Phosphate, Monobasic 0.93g 0.93 g 0.93 g Sodium Chloride 0.2 g 0.2 g 0.2 g Water up to 100 ml 100ml 100 ml

Calculated amounts of drug(s), vitamin E TPGS and octoxynol-40 requiredfor 10 mL were weighed, then mixed in 4 mL 95% ethanol, and evaporatedunder vacuum to form a thin film near-solid matter. The thin filmnear-solid matter was then dissolved in 5 mL deionized water andsonicated approximately 40 minutes to ensure complete formation of mixedmicelles. The prepared basic formulations were stored at roomtemperature.

A buffer mixture containing sodium phosphate, dibasic, sodium phosphate,monobasic and sodium chloride was prepared by dissolving in deionizedwater. Stock solution PVP-K-90 was prepared in water. The requiredvolume of polymer solution and buffer solution was added to the basicformulations and gently vortexed to get a clear solution. The pH of thesolution was adjusted with NaOH or HCl to a target of about 6.8. Theformulation was sterilized by a nylon membrane filter (0.22 μm) and thenstored at room temperature until use. The micellar size of formulationswas measured by using dynamic light scattering technique (Brookhaven90Plus particle size analyzer, Holtsville, N.Y.), taking the average ofthree measurements. The results of the study are described below. Theformulations were found to be clear and transparent at room temperature.The micellar size and polydispersity (PDI) index of the formulations aregiven in Table 34.

TABLE 34 Observed micelle size and PDI of the formulations Formulationcontaining Micelle Size (nm) PDI Cyclosporine 12.6 ± 0.2 0.119 ± 0.004Sirolimus 13.9 ± 0.1 0.198 ± 0.002 Tacrolimus 13.8 ± 0.2 0.199 ± 0.005

Example 16. Artificial Tear Compositions

TABLE 35 Biocompatible Artificial Tear Composition Ingredient AmountVoclosporin 0 Vitamin E TPGS 2.5 g Octoxynol-40 2.0 g PVP-K-90 1.2 gSodium Phosphate, Dibasic 0.81 g Sodium Phosphate, 0.93 g MonobasicSodium Chloride 0.2 g Water up to 100 mL

To show that none of the components of the artificial tear compositionsof the present disclosure are inherently irritating to ocular tissues, astudy was performed to determine ocular tolerability and toxicity of theartificial tears.

New Zealand White (NZW) rabbits (5 female/5 male) were topicallyadministered one approximately 35 μl drop or the artificial tearcomposition of the present disclosure to each eye at 1 hour intervals,for a maximum of up to 8 times per day. Animals were sacrificedfollowing 14 days of artificial tear administration. The followingparameters were evaluated during the study: morbidity/mortality,physical examination, clinical observations, body weights, feedconsumption, macro- and microscopic ocular observations,electroretinography (ERG), intraocular pressure measurement (IOP), andupon necropsy, histopathology was performed on the following tissues:eyes, thymus, mandibular, rostral and caudal lymph nodes, spleen. Allanimals were healthy and showed no findings outside the normal range.Eye related examination reports are provided in further details below:

Microscopic Ocular Grading:

The microscopic ocular grading system was applied to ocular findingsfollowing use of the slit lamp biomicroscope which included insertion ofa blue filter to assess for fluorescein dye retention. No lesions werenoted by indirect ophthalmoscopy performed pre-dose, and after 14 daysof artificial tear composition application (8 times per day).

Tonometry (IOP) Data Observations:

Mean tonometry (Tono-pen) readings of intraocular pressures (IOPs) inrabbits performed pre-test and after 14 days of artificial tearcomposition application were between 11-17 mm/Hg pressure and werewithin the normal physiologic range (10-20/mm Hg). In conclusion, no IOPeffects were observed in association with topical treatmentsadministered (8 times per day).

ERG Data Observations:

Bilateral full-field flash ERGs were performed in rabbits utilizing theISCEV protocol and the HMsERG unit. Preliminary evaluation of maximum a-and b-wave amplitudes for high intensity stimulation with 10 cd.s/m2,and 30 Hz flicker stimulation, also using 10 cd.s/m2, both underscotopic conditions, did not show any findings after 14 days ofartificial tear composition application (8 times per day).

Histopathology Observations

There were no histopathologic findings after 14 days of artificial tearcomposition application (8 times per day)

Example 17. Mixed Micellar Formulations Containing Sugar Additives

Sugar additives, such as trehalose, mannose, D-galactose and lactosewere added to the various formulations of the present disclosure andstability studies were carried out at different temperatures. Sugarswere added to the formulations during the rehydration step (externally),or added prior to the creation of the thin-film (internally). Theformulations were found to be stable in the presence of the adjuvantsugars.

Formulations containing decreased concentration of octoxynol-40 withsugar were also prepared where sugar was added during the preparation ofbasic formulation (internally). Studies were carried out with 0.05% and0.1% octoxynol-40 and 0.5% and 1.0% sugar for stability studies. Theresults obtained during the studies showed that the formulation remainedstable until 35 days at 30° C.

TABLE 36 Compositions of formulations (sugars added internally).Voclosporin 0.2% 0.2% 0.2% 0.2% Vitamin E 2.5 2.5 2.5 2.5 TPGSOctoxynol- 0.05%  0.1% 0.05%  0.1% 40 Trehalose 0.5% 0.5% 1.0% 1.0%Water up to 100 ml 100 ml 100 ml 100 mlMethod:

Calculated amounts of drug (about 0.2%, i.e., 200 mg), vitamin E TPGS(about 2.5%, i.e., 2.5 g) and octoxynol-40 (about 0.05/0.1%, i.e.,50/100 mg) required for 100 mL of the formulation were weighed. Twohundred milligrams of drug, about 2.5 g of TPGS and about 50/100 mg ofoctoxynol-40 were dissolved in about 2 ml, about 1 ml and about 50/100μL of 95% ethanol, respectively. For sugar, about 1 g of trehalose wasdissolved in about 4.5 ml of water/ethanol mixture (about 2.5 mlwater+about 2.0 ml ethanol) separately and mixed with other contents.Same water:ethanol ratio was used for preparing formulations containingdifferent amounts of sugar. The mixture was then evaporated under vacuumovernight to form a thin film. The thin film was then dissolved in about45 mL deionized water and sonicated for approximately 45 min to ensurecomplete formation of mixed micelles.

The rehydrating solution containing sodium phosphate, dibasic (about0.8092%), sodium phosphate, monobasic (about 0.9287%), sodium chloride(about 0.18%) and the polymer PVP-K 90 (about 1.2%) was prepared bydissolving amounts in about 45 mL of deionized water. This polymersolution was then added to the previously prepared micelles in ameasuring cylinder and the volume was made up to about 100 mL withdeionized water (q.s.). Finally the pH of the formulation was adjustedwith NaOH or HCl to about 6.8. The formulation was sterilized by a nylonmembrane filter (0.22 rpm).

During stability studies, at predetermined time intervals samples werewithdrawn, centrifuged and the supernatant solution was collected foranalysis of drug content.

Results:

The formulations were found to be clear and transparent at roomtemperature before the start of stability studies. Micellar sizeobserved was in the range of 12-14 nm. Example formulations withoctoxynol-40 and trehalose are as follows:

TABLE 37A Formulations with sugar additives. Code Formulation Label B0.05% OC-40 + 0.5% trehalose C 0.1% OC-40 + 0.5% trehalose E 0.05%OC-40 + 1.0% trehalose F 0.1% OC-40 + 1.0% trehalose

TABLE 37B Percentage drug remaining of different formulations at 30° C.Day 0 2 4 6 8 13 18 25 35 B 100.00 103.11 98.26 98.20 98.72 101.59 98.85107.14 95.40 C 100.00 98.43 94.55 95.50 96.66 98.65 94.88 93.84 95.21 E100.00 96.37 97.22 99.04 97.61 99.38 95.88 93.12 91.75 F 100.00 99.5498.33 100.33 99.00 100.97 95.11 95.79 97.27

Example 18. Ocular Distribution and Pharmacokinetics of 0.2 wt %/Vol.Voclosporin in Mixed Micellar Formulations of the Present Disclosure

The purpose of this study was to assess the temporal distribution andpotential accumulation with repeat dosing, gender difference, andpotential melanin binding of a 0.2% ¹⁴C-radiolabeled voclosporincomposition (ophthalmic solution) of the present disclosure after ocularapplication by determining radioactivity in ocular tissues, tears, andblood in New Zealand White (NZW) and Dutch Belted (DB) rabbits.

Methods:

NZW rabbits (30 females/8 males) were used in a single dose (SD) and7-day repeat dose (RD) study (see Table 38). DB rabbits (16 females)were used in a single dose study (see Table 39). Animals were either nottreated (controls) or given a single or a daily topical ocular dose for7 days (35 μL of 0.2% ¹⁴C-voclosporin in a mixed micellar formulation toone or both eyes). Blood and ocular tissue radioactivity levels wereassessed at designated time points via combustion followed by liquidscintillation counting. No mortality, morbidity or evidence of clinicalirritation occurred in any of the rabbits.

TABLE 38 Ocular Tissue Distribution of ¹⁴C-Voclosporin in Mixed MicellarComposition. Group No. of ¹⁴C-Dose Sample Collection Time IDAnimals/group Administration^(a) Matrices Collected (Time of euthanasia)1^(b)  2 ♀ None Tear, Blood, Ocular Pre-dose  2 ♂ Tissues/Fluids 2^(c)12 ♀ 35 μL/eye, once, Tear, Blood, Ocular ♀: 0.5, 1, 2, 4, 8, and 24 hr 6 ♂ Ocular (bilateral) Tissues/Fluids ♂: 1, 4, and 24 hr (SD group)After the dose administration (2 animals/time point) 3  2 ♀ 35 μL/eye,once, Tear, Blood, Ocular 1 hr after the dose administration Ocular(unilateral) Tissues/Fluids 4^(d)  2 ♀ 35 μL/eye, once Tear, BloodOcular Just prior to 7^(th) dose administration in daily, bilateral forTissues/Fluids the next group 6 days 5^(e) 12 ♀ 35 μL/eye, once Tear,Blood Ocular 0.5, 1, 2, 4, 8, and 24 hr after the last daily, bilateralfor Tissues/Fluids dose administration 7 days (RD group) (2 animals/timepoint) ^(a)The topical dose formulation contained 0.2% voclosporin. Thetarget dose was ~3 μCi/35 μL and 70 ng voclosporin. ^(b)Used as predoseconcentration for Treatment Group 2 (SD group). ^(c)Used forpharmacokinetic assessment (SD group). ^(d)Used as predose concentrationfor Treatment Group 5 (RD group). ^(e)Used for pharmacokineticassessment (MD group).

TABLE 39 Ocular Tissue Distribution of ¹⁴C-voclosporin in Mixed MicellarComposition Group No. of ¹⁴C-Dose Sample Collection Time IDAnimals/group Administration ^(a) Matrices Collected (Time ofEuthanasia) 1^(b) 2 ♀ None Tear, Blood, Ocular Pre-dose Tissues/Fluids2^(c) 12 ♀  35 μL/eye, once, Tear, Blood, Ocular 0.5, 1, 2, 4, 8, and 24hr after the Ocular (bilateral) Tissues/Fluids dose administration (SDgroup) (2 animals/time point) 3 2 ♀ 35 μL/eye, once, Tear, Blood, Ocular1 hr after dose administration Ocular (unilateral) Tissues/Fluids ^(a)The topical dose formulation contained 0.2% voclosporin. The target dosewas ~3 μCi/35 μL and 70 ng voclosporin/dose. ^(b)Used as predoseconcentration for Treatment Group 2 (SD group). ^(c)Used forpharmacokinetic assessment (SD group).

At each sampling point, a t-test was used to compare the tissueconcentrations within or between the two strains of rabbits. SigmaStat®3.5 (Systat, Inc., San Jose, Calif.) was used for the statisticalanalyses (p<0.05). Non-compartmental analysis was performed on the meantissue ¹⁴C-voclosporin concentration—time data. Pharmacokinetic analysiswas performed using WinNonlin 5.2 (Pharsight, Corporation, MountainView, Calif.). C_(max) and T_(max), and where calculable AUC andt_(1/2), were reported.

Pharmacokinetic Parameters:

Selected pharmacokinetic parameters (C_(max), AUC, T_(max), and t_(1/2))for ¹⁴C-voclosporin-derived radioactivity are summarized in Tables 40and 41 for NZW and DB rabbits, respectively. After a single dose, therewas rapid penetration of drug (measured as radioactivity) into oculartissues with the highest concentrations (>1 mg eq/g tissue) occurring inthe eyelids, conjunctiva, cornea, nictitating membrane and tears, andthe lowest concentrations (1-11 ng eq/g tissue) in the aqueous andvitreous humor, and the lens. The remaining ocular tissues achievedvarious levels (20-223 ng eq/g tissue) of voclosporin and/or relatedresidue. FIG. 2 shows the tissue levels of ¹⁴C-voclosporin after asingle (1 day) topical dose of the 0.2% ¹⁴C-voclosporin mixed micellarformulation to female New Zealand White Rabbits. Therapeutic levels ofvoclosporin were noticed at the 24-hour mark, supporting once daily (QD)dosing is possible with the aqueous mixed micellar composition of thepresently disclosed embodiments.

Following repeat dosing of up to 7 days, based on limited availableinformation generated in this study (lower bulbar conjunctiva,nictitating membrane, and upper bulbar conjunctiva), there was noapparent change in ¹⁴C-voclosporin t_(1/2) (see Table 40). All but oneblood sample were below the lower limit of quantification (LLOQ) (3.06ng eq/mL) in the radioactivity assay. Notably, single doseadministration resulted in therapeutic levels (higher than 10 ngequivalent drug/gram tissue) in all ocular tissues (with the exceptionof aqueous/vitreous humor and lens), with negligible systemic exposure.

TABLE 40 Pharmacokinetic Parameters of ¹⁴C-voclosporin-derivedradioactivity following a single or repeat (QD for 7 days), bilateralocular administration of ¹⁴C-voclosporin in a mixed micellar formulationto female NZW rabbits. Ocular T_(max) t_(1/2) Tissue(s)/Fluids C_(max)(ng eq./g) AUC (hr*ng eq./g) (hr) (hr) & Blood SD RD Ratio SD RD RatioSD RD SD RD Aqueous Humor 6 13 2.3 45 96 2.1 0.5 0.5 — 14 Choroid/Retina 48 76 1.6 472 897 1.9 1.0 2.0 23  — Cornea 1203 3382 2.823166 54624 2.4 8.0 0.5 — — Iris/Ciliary Body 20 119 5.8 382 1952 5.124.0 1.0 — — Lacrimal Gland 31 120 3.9 416 1109 2.7 2.0 4.0 — 6 Lens 426 6.7 47 356 7.5 24.0 0.5 — — Lower Bulbar 1810 2929 1.6 12029 165851.4 0.5 0.5 10  7 Conjunctiva Lower Eyelid 20814 41635 2.0 207630 3587911.7 1.0 0.5 — — Nictitating 1716 2468 1.4 12135 15964 1.3 0.5 0.5 7 8Membrane Optic Nerve 83 164 2.0 569 1805 3.2 0.5 0.5 — 16  Sclera 223367 1.6 2646 3825 1.4 0.5 0.5 — 16  Submandibular 74 120 1.6 893 11901.3 2.0 2.0 — — Lymph Node Tear 20246 30904 1.5 168259 230878 1.4 0.50.5 — 7 Upper Bulbar 2235 3170 1.4 14782 19944 1.3 0.5 0.5 7 7Conjunctiva Upper Eyelid 9896 17500 1.8 114651 98656 0.9 1.0 0.5 — 4Vitreous Humor 2 2 1 27 23 0.9 8.0 4.0 — — Blood BQL BQL NC NC NC NC NCNC NC NC SD = Single dose; RD = Repeat Dose; Ratio = Repeat Dose/SingleDose.; — = Insufficient tissue concentrations to determine t_(1/2); BOL= Below Quantifiable Limit (<0.1 ng/mL); NC = Not calculated.

TABLE 41 Pharmacokinetic Parameters of ¹⁴C-voclosporin-derivedradioactivity following a single bilateral ocular administration of¹⁴C-voclosporin in a mixed micellar formulation according to the presentdisclosure to female DB Rabbits. Ocular Tissue(s)/ C_(max) T_(max)t_(1/2) AUC Fluids & Blood (ng eq./g) (hr) (hr) (hr*ng eq./g) AqueousHumor 11 0.5 — 56 Choroid/Retina 49 1.0 — 92 Cornea 1519 8.0 — 27844Iris/Ciliary Body 30 24.0 — 541 Lacrimal Gland 75 1.0 — 335 Lens 2 24.0— 26 Lower Bulbar Conjunctiva 2080 1.0 15 13107 Lower Eyelid 69055 4.0 —512473 Nictitating Membrane 2400 1.0 12 13091 Optic Nerve 192 1.0 161127 Sclera 220 1.0 — 3502 Submandibular Lymph Node 86 4.0 — 635 Tear57476 1.0 — 262299 Upper Bulbar Conjunctiva 2491 1.0 14 14296 UpperEyelid 8245 4.0 — 68063 Vitreous Humor 1 1.0 — 16 Blood BQL NC NC NC

TABLE 42 Comparative C_(max) of ¹⁴C-voclosporin derived radioactivity inNZW and DB rabbits after single topical ocular administration of¹⁴C-voclosporin. New Zealand White Dutch Belted (Study No. S08861)(Study No. S08862) Ocular Tissue(s)/Fluids C_(max) C_(max) & Blood (ngeq./g) (ng eq./g) Aqueous humor 6 11 Choroid/Retina 48 49 Cornea 12031519 Iris/Ciliary Body 20 30 Lacrimal Gland 31 75 Lens 4 2 Lower BulbarConjunctiva 1810 2080 Lower Eyelid 20814 69055 Nictitating membrane 17162400 Optic Nerve 83 192 Sclera 223 220 Submandibular Lymph Node 74 86Tear 20246 57476 Upper Bulbar Conjunctiva 2235 2491 Upper Eyelid 98968245 Vitreous Humor 2 1 Blood BQL BQL

TABLE 43 Ocular tissues/fluids distribution (C_(max)) of ¹⁴C-voclosporinin NZW Rabbits. ¹⁴C-voclosporin (0.2%, ¹⁴C-voclosporin aqueous solution)Once a day (QD) Ocular Single dose 7 Days Tissue(s)/Fluids C_(max)C_(max) & Blood (ng eq./g)^(a) (ng eq./g)^(a) Aqueous humor 6 13Choroid/Retina 48 76 Cornea 1203 3382 Iris/Ciliary Body 20 119 LacrimalGland 31 120 Lens 4 26 Lower Conjunctiva 1810 2929 Lower Eyelid 2081441635 Nictitating membrane 1716 2468 Optic Nerve 83 164 Sclera 223 367Submandibular Lymph 74 120 Node Tear 20246 30904 Upper Conjunctiva 22353170 Upper Eyelid 9896 17500 Vitreous Humor 2 2 Blood BQL BQL

FIGS. 3A-D show mean ocular tissue concentrations of ¹⁴C-voclosporinafter a single (1 day) or repeat (7 days), bilateral, once daily,topical dose of the 0.2% ¹⁴C-voclosporin mixed micellar formulation tofemale New Zealand White Rabbits (FIG. 3A, cornea; FIG. 3B, iris/ciliarybody; FIG. 3C, lacrimal gland; and FIG. 3D, lens).

FIGS. 4A-D show mean ocular tissue concentrations of ¹⁴C-voclosporinafter a single (1 day) or repeat (7 days), bilateral, once daily,topical dose of the 0.2% ¹⁴C-voclosporin mixed micellar formulation tofemale New Zealand White Rabbits (FIG. 4A, lower conjunctiva; FIG. 4B,lower eyelid; FIG. 4C, nictitating membrane; and FIG. 4D, sclera).

FIGS. 5A-D show mean ocular tissue and fluid concentrations of¹⁴C-voclosporin after a single (1 day) or repeat (7 days), bilateral,once daily, topical dose of the 0.2% ¹⁴C-voclosporin mixed micellarformulation to female New Zealand White Rabbits (FIG. 5A, upperconjunctiva; FIG. 5B, upper eyelid; FIG. 5C, aqueous humor; and FIG. 5D,vitreous humor).

FIGS. 6A-D show mean ocular tissue and fluid concentrations of¹⁴C-voclosporin after a single (1 day) or repeat (7 days), bilateral,once daily, topical dose of the 0.2% ¹⁴C-voclosporin mixed micellarformulation to female New Zealand White Rabbits (FIG. 6A, tears; FIG.6B, lymph node; FIG. 6C, optic nerve; and FIG. 6D, choroid/retina).

FIG. 7 is a graph showing C_(max) values of ¹⁴C-voclosporin after repeat(7 day), bilateral, once daily, topical dose of the 0.2% ¹⁴C-voclosporinmixed micellar formulation to female New Zealand White Rabbits.

Potential Accumulation of ¹⁴C-Voclosporin-Derived Radioactivity:

Ocular exposure to ¹⁴C-voclosporin ocular exposure was increased 2.8 to6.7 fold in cornea, lacrimal gland, iris/ciliary body and lens after 7days of once daily, bilateral ocular administration of ¹⁴C-voclosporin(35 μL, 70 ng) (see Table 40). After multiple dosing (see Tables 40-43and FIGS. 3-7), even though the C_(max)-repeat dose:C_(max)-single doseratio was elevated in selected tissues, the overall levels ofvoclosporin were well below the surface tissue levels indicating minimaltissue accumulation. Also, comparable t_(1/2) after single or repeatdosing strongly suggested minimal tissue accumulation.

Potential for Melanin Binding:

Following a single dose of ¹⁴C-voclosporin to DB rabbits, ocular tissueconcentrations (e.g., C_(max)) were not significantly different from NZWrabbits, suggesting a lack of melanin binding (see Table 42).

High levels of drug are achievable with one topical application (singledose) of the compositions of the present disclosure. More particularly,high drug levels were maintained in ocular tissues for up to, andbeyond, 24 hours post-administration, suggesting that QD (once daily)dosing is achievable using the compositions of the present disclosure.The concentration of drug is high in tissues in the front of the eye(cornea, conjunctiva, sclera) and at the back of the eye (retina, opticnerve) but minimal in the middle of the eye (aqueous and vitreoushumor), suggesting transport of the drug by a mechanism other thanpassive transport through the eye. The high drug levels achieved at theback of the eye make topical administration of the compositions of thepresent disclosure feasible for the treatment of diseases of theback-of-the-eye (e.g., retinal, diseases involving optic nerve such asglaucoma). Various water-insoluble drugs can be used with thecompositions of the present disclosure, including, but not limited to,calcineurin and mTOR inhibitors. Very high levels, especially in targettissues such as lachrymal gland, have been shown with the compositionsof the present disclosure.

Concentrations of ¹⁴C-voclosporin-derived radioactivity (ng eq/g tissue)that exceeded therapeutic levels (>10 ng eq/g tissue) were measured inall ocular tissues except in the lens and ocular fluids (aqueous humor,vitreous humor) after single and repeat ocular applications. Bloodlevels were at the lower limit of quantification (LLOQ) suggestingminimal systemic exposure, and there was minimal distribution of¹⁴C-voclosoporin to the contralateral, non-treated eye, likely due tothe grooming behavior of animals.

Ocular exposure to ¹⁴C-voclosporin in the mixed micellar formulation ofthe present disclosure, as demonstrated by C_(max) and AUC, variedwidely among the ocular tissues. ¹⁴C-voclosporin exposure was highest inthe ocular adnexa and exterior tissues (cornea, sclera, lower bulbarconjunctiva, lower eyelid, nictitating membrane, upper bulbarconjunctiva and upper eyelid) and tears, and lowest in the interiorocular tissues and fluids (vitreous humor, lens, aqueous humor); and inthe middle range in the iris/ciliary body, lacrimal gland, submandibularlymph nodes, choroid/retina and optic nerve. Most ocular tissue levelsthus exceed the 10 ng eq/g level needed for the biologic effect.

After once a day, daily ocular applications of ¹⁴C-voclosporin in amixed micellar formulation for 7 days, concentrations of ¹⁴C-voclosporinin target tissues (e.g., conjunctiva, cornea, and lacrimal gland)remained at therapeutic levels even at the 24-hour mark, supporting oncedaily (QD) dosing is possible with an aqueous mixed micellar compositionof the presently disclosed embodiments.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. It will beappreciated that various of the above-disclosed and other features andfunctions, or alternatives thereof, may be desirably combined into manyother different systems or applications. Various presently unforeseen orunanticipated alternatives, modifications, variations, or improvementstherein may be subsequently made by those skilled in the art which arealso intended to be encompassed by the following claims.

What is claimed is:
 1. A pharmaceutical composition comprising: acyclosporine; a first surfactant with an HLB index greater than about10; and a second surfactant with an HLB index greater than about 13,wherein the first surfactant is a polyethylene glycol (PEG)-5-100 nonylphenyl ether or a PEG-glycerol fatty acid ester, the second surfactantis octoxynol-40, and an absolute difference between the HLB index of thefirst surfactant and the HLB index of the second surfactant is greaterthan about 3, wherein the pharmaceutical composition is in the form ofmixed micelles having the first and second surfactants, and wherein thepharmaceutical composition contains less than 2% by weight ethanol. 2.The pharmaceutical composition of claim 1 wherein the cyclosporine iscyclosporine A.
 3. The pharmaceutical composition of claim 1 or 2wherein the first surfactant is a PEG-glycerol fatty acid ester.
 4. Thepharmaceutical composition of claim 3 wherein the pharmaceuticalcomposition is optically clear.
 5. The pharmaceutical composition ofclaim 3 wherein the cyclosporine is able to reach the back of an eyeafter topical administration to the eye.
 6. The pharmaceuticalcomposition of claim 3 wherein the pharmaceutical composition is free ofshort-chain alcohols.
 7. The pharmaceutical composition of claim 3wherein the pharmaceutical composition is an aqueous solution.
 8. Amethod for treating an ocular disease, comprising administeringtopically to an eye of a patient in need thereof a pharmaceuticalcomposition comprising: a cyclosporine; a first surfactant with an HLBindex greater than about 10; and a second surfactant with an HLB indexgreater than about 13, wherein the first surfactant is a polyethyleneglycol (PEG)-5-100 nonyl phenyl ether or a PEG-glycerol fatty acidester, the second surfactant is octoxynol-40, and an absolute differencebetween the HLB index of the first surfactant and the HLB index of thesecond surfactant is greater than about 3, wherein the pharmaceuticalcomposition is in the form of mixed micelles having the first and secondsurfactants, and wherein the pharmaceutical composition contains lessthan 2% by weight ethanol.
 9. The method of claim 8 wherein thecyclosporine is cyclosporine A.
 10. The method of claim 8 or 9 whereinthe first surfactant is a PEG-glycerol fatty acid ester.
 11. The methodof claim 10 wherein the pharmaceutical composition is optically clear.12. The method of claim 10 wherein the pharmaceutical composition isfree of short-chain alcohols.
 13. The method of claim 10 wherein thepharmaceutical composition is an aqueous solution.
 14. The method ofclaim 10 wherein the cyclosporine is delivered to back of the eye viathe pharmaceutical composition.
 15. The method of claim 10 wherein theocular disease is keratoconjunctivitis sicca (KCS).